Hormone receptor-related bioassays

ABSTRACT

The present invention discloses two hormone receptor-related bioassays. The first bioassay is useful for determining whether a protein suspected of being a hormone receptor has transcription-activating properties of a hormone receptor. The second bioassay is useful for evaluating whether compounds are functional ligands for receptor proteins. According to the first bioassay, cells that contain non-endogenous DNA which expresses a protein suspected of being a hormone receptor and which contain a DNA sequence encoding an operative hormone responsive promoter/enhancer element linked to an operative reporter gene, are cultured, the culturing being conducted in a culture medium containing a known hormone, or an analog thereof. The cultured cells are then monitored for induction of the product of the reporter gene as an indication of functional transcription-activating binding between the hormone or hormone analog and the protein suspected of being a hormone receptor. According to the second bioassay, cells that contain non-endogenous DNA which expresses hormone receptor or a functional engineered or modified form thereof, and which also contain a DNA sequence encoding an operative hormone responsive promoter/enhancer element linked to an operative reporter gene, are cultured, the culturing being conducted in culture medium containing at least one compound whose ability to functionally bind the receptor protein is sought to be determined. The cultured cells are then monitored for induction of the product of the report gene as an indicator of functional binding between the compound and the receptor.

ACKNOWLEDGMENT

This invention was made with government support under a grant from theNational Institutes of Health (Grant No. GM 26444).

RELATED APPLICATIONS

This is a continuation-in-part of U.S. Ser. No. 922,585, filed Oct. 24,1986 now abandoned.

FIELD OF THE INVENTION

The present invention relates to hormone receptor proteins and genesencoding them, modification of such receptors and genes by recombinantDNA and other genetic engineering techniques, plus uses of suchreceptors and genes, both unmodified and modified. More particularly,the invention concerns steroid and thyroid hormone receptors andassociated genes. Most particularly, it concerns human glucocorticoid,mineralocorticoid and thyroid hormone receptors and genes for them. Inaddition the invention relates to a novel bioassay system fordetermining the functionality of hormone receptor proteins coded for byreceptor DNA clones, plus novel methods for inducing and controllingexpression of genes whose transcription is activated by hormonescomplexed with receptor proteins.

BACKGROUND OF THE INVENTION

Transcriptional regulation of development and homeostasis in complexeukaryotes, including humans and other mammals, birds, and fish, iscontrolled by a wide variety of regulatory substances, including steroidand thyroid hormones. These hormones exert potent effects on developmentand differentiation in phylogenetically diverse organisms and theiractions are mediated as a consequence of their interactions withspecific, high affinity binding proteins referred to as receptors. Seegenerally, Jensen, et al., (1972); Gorski, et al., (1976); Yamamoto, etal., (1976); O'Malley, et al., (1969); Hayward, et al., (1982); andAsburner, et al., (1978).

Receptor proteins, each especially specific for one of the severalclasses of cognate steroid hormones (i.e., estrogens (estrogenreceptor), progestogens (progesterone receptor), glucocorticoids(glucocorticoid receptor), androgens (androgen receptor), aldosterones(mineralocorticoid receptor) or for cognate thyroid hormones (thyroidhormone receptor), are known and distributed in a tissue specificfashion. See Horwitz, et al., (1978) and Pamiter, et al., (1976).

Turning now to the interaction of hormones and receptors, it is knownthat a steroid or thyroid hormone enters cells by facilitated diffusionand binds to its specific receptor protein, initiating an alostericalteration of the protein. As a result of this alteration, thehormone/receptor complex is capable of binding to certain specific siteson chromatin with high affinity. See Yamamoto, et al., (1972) andJensen, et al., (1968).

It is also known that many of the primary effects of steroid and thyroidhormones involve increased transcription of a subset of genes inspecific cell types. See Peterkofsky, et al., (1968) and McKnight, etal., (1968). Moreover, there is evidence that activation oftranscription (and, consequently, increased expression) of genes whichare responsive to steroid and thyroid hormones (through interaction ofchromatin with hormone receptor/hormone complex) is effected throughbinding of the complex to enhancers associated with the genes. (SeeKhoury, et al., 1983.)

In any case, a number of steroid hormone and thyroid hormone responsivetranscriptional control units, some of which have been shown to includeenhancers, have been identified. These include the mouse mammary tumorvirus 5'-long terminal repeat (MTV LTR), responsive to glucocorticoid,aldosterone and androgen hormones; the transcriptional control units formammalian growth hormone genes, responsive to glucocorticoids,estrogens, and thyroid hormones; the transcriptional control units formammalian prolactin genes and progesterone receptor genes, responsive toestrogens; the transcriptional control units for avian ovalbumin genes,responsive to progesterones; mammalian metallothionein genetranscriptional control units, responsive to glucocorticoids; andmammalian hepatic alpha_(2u) -globulin gene transcriptional controlunits, responsive to androgens, estrogens, thyroid hormones andglucocorticoids. (See the Introduction portion of Experimental Section Iof this Specification for references.)

A major obstacle to further understanding and more practical use of thesteroid and thyroid hormone receptors has been the lack of availabilityof the receptor proteins, in sufficient quantity and sufficiently pureform, to allow them to be adequately characterized. The same is true forthe DNA gene segments which encode them. Lack of availability of theseDNA segments has prevented in vitro manipulation and in vivo expressionof the receptor-coding genes, and consequently the knowledge suchmanipulation and expression will yield.

The present invention is directed to overcoming these problems of shortsupply of adequately pure receptor material and lack of DNA segmentswhich encode the receptors.

REFERENCE LIST

The Background section of the specification refers to the followingpublications.

PUBLICATIONS

1. Asburner, M., and Berendes, H. D. in The Genetics and Biology ofDrosophila, Eds. Ashburner, M., and Wright, T. R. F., Vol. 2, pp.315-395, Academic, London (1978).

2. Gorski, J., and Gannon, F., A. Rev. Physiol., 38:425-450 (1976).

3. Hayward, M. A., Brock, M. L. and Shapiro, D. J., Nucleic Acids Res.,10:8273-8284 (1982).

4. Horwitz K. B. and McGuire W. L. J. Biol. Chem., 253:2223-2228 (1978).

5. Jensen, E. V., and DeSombre, E. R., A. Rev. Biochem., 41:203-230(1972).

6. Jensen, E. V., et al., Proc. Natl. Acad. Sci. U.S.A., 59:632-638(1968).

7. Khoury, G., and Gruss, P., Cell, 33:313-314 (1983).

8. McKnight, G. S., and Palmiter, R. D., J. Biol. Chem., 254:9050-9058(1968).

9. O'Malley, B. W., McGuire, W. L., Kohler, P. O., and Kornman, S. G.,Recent Prog. Horm. Res., 25:105-160 (1969).

10. Pamiter, R. D., Moore, P. B., Mulvihill, E. R. and Emtage, S., Cell,8:557-572 (1976).

11. Peterkofsky, B., and Tomkins, G., Proc. Natl. Acad. Sci. U.S.A.60:222-228 (1968).

12. Yamamoto, K. R., and Alberts, B. M., A. Rev Biochem., 45:721-746(1976).

13. Yamamoto, K. R., and Alberts, B. M., Proc. Natl. Acad. Sci. U.S.A.69:2105-2109 (1972).

OTHER PUBLICATIONS

Some of the information disclosed in this specification has beenpublished:

The study disclosed in Experimental Section I has been published as:Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A.,Lebo, R., Thompson, E. B., Rosenfeld, M. G., and Evans, R. M., "PrimaryStructure and Expression of a Functional Human Glucocorticoid ReceptorcDNA", Nature (London), 318:635-641 (December, 1985).

The study disclosed in Experimental Section II has been published as:Giguere, V., Hollenberg, S. M., Rosenfield, M. G., and Evans, R. M.,"Functional Domains of the Human Glucocorticoid Receptor", Cell,46:645-652 (August, 1986).

The study disclosed in Experimental Section III has been published as:Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruol, D. J., andEvans, R. M., "The c-erb-A Gene Encodes a Thyroid Hormone Receptor",Nature (London), 324:641-646 (December, 1986).

The study disclosed in Experimental Section IV has been published as:Arriza, J. L., Weinberger, C., Cerelli, G., Glaser, T. M., Handelin, B.L., Houseman, D. E., and Evans, R. M., "Cloning of HumanMineralocorticoid Receptor Complementary DNA: Structural and FunctionalKinship with the Glucocorticoid Receptor", Science 237:268-275 (July,1987).

The study disclosed in Experimental Section V is in press as: Giguere,V., Yang, N., Segui, P., and Evans, R. M., "Identification of a NewClass of Steroid Hormone Receptors".

The study disclosed in Experimental Section VI is in press as: Glass, C.K., Franco, R., Weinberger, C., Albert, V. R., Evans, R. M., andRosenfeld, M. G., "A c-erb-A Binding Site in the Rat Growth Hormone GeneMediates Transactions by Thyroid Hormone".

The study disclosed in Experimental Section VII has been published as:Thompson, Catherine C., Weinberger, Cary, Lebo, Roger, and Evans, RonaldM., "Identification of a Novel Thyroid Hormone Receptor Expressed in theMammalian Central Nervous System", Science, 237:1610-1614 (September,1987).

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings. More detaileddescriptions are found in the Experimental Sections of thisspecification. To avoid confusion, drawings which are referred to inExperimental Section I are labeled with the prefix "I"; those referredto in Experimental Section II are labeled with the prefix "II", and soon.

The drawings comprise 40 Figures, of which:

EXPERIMENTAL SECTION I

FIG. I-1 (a and b ) is a drawing which shows the human glucocorticoidreceptor cDNA sequencing strategy (FIG. I-1a), plus a schematicrepresentation of cDNA clones (FIG. I-1b).

FIG. I-2 (parts A to H) is a drawing which shows the cDNA and predictedprimary protein sequence of human glucocorticoid receptor (hGR).

FIG. I-3 (a and b) is a drawing which shows the restriction map (FIG.I-3a) and nucleotide sequence (FIG. I-3b) of the 3' end of the humanglucocorticoid receptor beta cDNA (beta-hGR).

FIG. I-4 (a and b) relates to an immunoblot comparison of hGR translatedin vitro with in vivo hGR from cell extracts. FIG. I-4a is a drawingshowing the vectors constructed for in vitro transcription of the hGRcDNA sequence. FIG. I-4b is a photograph showing a Western blot analysisof in vitro translation products and cell extracts.

FIG. I-5 is a graph showing steroid-binding of alpha-hGR (GR107)translated in vitro.

FIG. I-6 (parts A and B) is a schematic drawing of expression plasmidspGERR1 and pGERR2. Plasmid pGERR1 was used to express estrogen relatedreceptor hERR1; pGERR2 was used to express hERR2.

EXPERIMENTAL SECTION II

FIG. II-1 is a drawing showing a schematic representation of the hGRfunctional assay.

FIG. II-2 is a photograph showing a Western blot analysis whichillustrates expression of hGR protein.

FIG. II-3 is a photograph of a blot which illustrates induction of CATactivity by hGR.

FIG. II-4 (A and B) is a graph. FIG. II-4A shows the dose-response toDEX of pRShGR alpha; FIG. II-4B shows the titration of pRShGRa.

FIG. II-5 is a schematic drawing showing the location of functionaldomains in hGR.

EXPERIMENTAL SECTION III

FIG. III-1 (parts A, B1 and B2) is a drawing which shows (a) therestriction map and sequencing strategy, plus (b) the nucleotide andpredicted amino acid sequence, of human placenta c-erb-A cDNA.

FIG. III-2 is a drawing showing an amino acid sequence comparisonbetween the carboxy-terminal portions of the v-erb-A oncogene product,the human placental c-erb-A polypeptide and the human glucocorticoid andoestrogen receptors.

FIG. III-3 (a, b and c) is a photograph of a blot showing Southernanalysis and chromosome mapping of human placental DNA with c-erb-A DNAprobes. FIG. III-3a shows human term placental DNA digested withendonucleases and separated on an agarose gel. FIG. III-3b shows ananalysis of a placental DNA using c-erb-A as a probe. FIG. III-c showschromosome mapping of the human c-erb-A genes.

FIG. III-4 (a and b) is a photograph showing human c-erb-A expression.FIG. III-4a is a blot showing a Northern analysis of RNA's from humancell lines and human placenta. FIG. III-4b illustrates synthesis oferb-A polypeptide in vitro.

FIG. III-5 (a, b, c and d) shows four graphs which relate to thyroidhormone binding to erb-A polypeptides synthesized in vitro. FIG. III-5ais a Scatchard analysis of ¹²⁵ I-T₃ binding to the erb-A polypeptidesmade in vitro. FIG. III-5b shows competition of thyroid hormoneanalogues in vitro. FIG. III-5c shows competition of triiodothyronineisomers from ¹²⁵ I-T₃ binding to erb-A polypeptides synthesized invitro. FIG. III-5d shows competition of thyroid hormone analogues for¹²⁵ I-T₃ binding to 0.4 KCl HeLa cell nuclear extracts.

FIG. III-6 is a schematic drawing which compares the steroid and thyroidhormone receptors.

FIG. III-7 (parts A and B) is a drawing which shows the cDNA nucleotidesequence and the predicted primary protein sequence of human thyroidreceptor hERBA 8.7. (The sequence of thyroid receptor hFA 8 is relatedto hERBA 8.7. See the Description of the Invention section whichfollows.)

EXPERIMENTAL SECTION IV

FIG. IV-1 (A, B, C and D) is comprised of three photographs and oneschematic drawing, all of which concern to isolation of a genomicsequence related to the hGR gene. FIG. IV-1A is a photograph showinghigh-stringency Southern analysis of human placenta DNA digested withthe indicated nucleases. FIG. IV-4B is similar except that it showslow-stringency Southern analysis. FIG. IV-4C is also a photograph of aSouthern blot; it demonstrates isolation of the genomic sequence in aclone designated lambda HGH. FIG. IV-4D is a schematic drawing whichshows the intron-exon structure of lambda HGH genomic fragment and itshomology with hGR.

FIG. IV-2 (parts A, Ba to Be) is a drawing which shows the cDNAnucleotide sequence and the predicted primary protein sequence of humanmineralocorticoid receptor. FIG. IV-2A shows the composite structure ofhMR aligned with a line diagram of some restriction endonucleasecleavage sites. FIG. IV-2B shows the complete nucleotide sequence of hMRand its primary predicted amino acid sequence.

FIG. IV-3 is a drawing which shows the amino acid homology betweenmineralocorticoid receptor and glucocorticoid receptor.

FIG. IV-4 (A, B, C and D) is a drawing and three graphs which relate tothe steroid-binding properties of expressed hMR. FIG. IV-4A shows thestructure of expression plasmid pRShMr, the plasmid used to express hMR.FIG. IV-4B is a graph showing Scatchard analysis of tritiatedaldosterone binding in extracts prepared from pRShMR-transfected COScells. FIGS. IV-4C and D are graphs showing competition of unlabeledsteroids for binding with [³ H]aldosterone in transfected COS cells.

FIG. IV-5 (A, B and C) is a drawing and two photographs which showtranscriptional activation of MMTV LTR by hMR and hGR expressionplasmids in transfected CV-1 cells. FIG. IV-5A is a schematic drawing ofplasmid GMCAT. FIG. IV-5B is a photograph of a blot which showsdifferential CAT enzyme activity found after hMR or hGR transfectionwith normal serum. FIG. IV-5C is a photograph of a blot which showsdifferential induction of CAT activity by aldosterone or dexamethasonein cells transfected with hMR or hGR.

FIG. IV-6 is a photograph of a blot showing Northern analysis ofmineralocorticoid receptor mRNA's in rat tissues.

FIG. IV-7 is a photograph showing chromosomal localization of hMR geneby Southern analysis of microcell hybrids.

FIG. IV-8 is a schematic drawing showing amino acid comparisons of thehGR, hMR, and hPR structures.

EXPERIMENTAL SECTION V

FIG. V-1 (parts A, Ba to Bd) is a drawing which shows the cDNAnucleotide sequence and the predicted primary protein sequence of hERR1.FIG. V-1A shows the composite structure of hERR1 aligned with a linediagram of some restriction endonuclease cleavage sites. FIG. V-1B showsthe complete nucleotide sequence of hERR1 and its primary predictedamino acid sequence.

FIG. V-2 (parts A, Ba to Be) is a drawing which shows the cDNAnucleotide sequence and the predicted primary protein sequence of hERR2.FIG. V-2A shows the composite structure of hERR2 aligned with a linediagram of some restriction endonuclease cleavage sites. FIG. V-2B showsthe complete nucleotide sequence of hERR2 and its primary predictedamino acid sequence.

FIG. V-3 is a drawing showing an amino acid sequence comparison betweenthe carboxy-terminal regions of hERR1, hERR2, the human oestrogen andglucocorticoid receptors.

FIG. V-4 is a photograph showing Northern blot hybridization analysis ofhERR1 (FIG. V-4A) and mRNA's in rat and human tissues (FIG. V-4B).

FIG. V-5 is a schematic drawing showing amino acid comparison betweenhERR1 and hERR2, hER and human thyroid hormone receptor (hT₃ R beta).

EXPERIMENTAL SECTION VI

FIG. VI-1 (a, b and c) is comprised of two drawings, plus a drawing anda photograph, all of which relate to thyroid hormone responsiveness ofvarious gene fusions containing rat GH 5'-flanking sequences. FIG. VI-1ais a drawing which illustrates responsiveness of 5' and 3' deletions ofthe rat GH gene. FIG. VI-1b is a drawing which shows functional analysisof the putative T₃ receptor binding site. FIG. VI-1c is adrawing/photograph combination which illustrates an mRNA transcriptioninitiation site analysis.

FIG. VI-2 (a and b) is a drawing which relates to binding of T₃receptors to oligonucleotide probes containing biotin-11-dUTP. FIG.VI-1a is a schematic representation of two oligonucleotide probes usedto assay T₃ receptor binding to GH 5'-flanking sequences. FIG. IV-1b isa graph showing precipitation of ¹²⁵ I-T₃ labeled T₃ receptors from GC2nuclear extracts by various oligonucleotide probes.

FIG. VI-3 is a photograph showing a DNase I footprinting of the rat GHenhancer element by GC2 nuclear extracts.

FIG. VI-4 is a graph illustrating binding to oligonucleotides containing64 and 29 base pairs of 5'-flanking GH sequence of rat pituitary cell T₃receptors and an hc-erb-A in vitro translation product.

EXPERIMENTAL SECTION VII

FIG. VII-1 (parts A, Ba, Bb) is a schematic drawing which shows therestriction map (A), plus the nucleotide and predicted amino acidsequence (B), of thyroid hormone receptor cDNA from rat brain clonerbeA12.

FIG. VII-2 is a schematic drawing which compares the rat thyroid hormonereceptor (rTR alpha) protein with the human thyroid hormone receptor(hTR beta) and chicken thyroid hormone receptor (cTR alpha) proteins.

FIG. VII-3 (A, B and C) is a photograph showing Southern blot analysisand human chromosomal localization of the rTR alpha gene. FIG. VII-3A isa blot showing human placenta DNA hybridized to a 500-bp PvuII fragmentfrom rbeA12; FIG. VII-3B shows the placenta DNA hybridized to a 450-bpSstI fragment from hTR beta. FIG. VII-C shows chromosome mapping of therTR alpha gene.

FIG. VII-4 (A, B and C) shows a photograph and two graphs which relateto in vitro translation and thyroid hormone binding of rTR alpha. FIG.VII-4A is a photograph of a SDS-polyacrylamide gel showing the in vitrotranslation products of rTR alpha. FIG. VII-4B is a graph showing aScatchard analysis of ¹²⁵ I-T₃ binding to in vitro translated rTR alpha.FIG. VII-4C is a graph showing competition of thyroid hormone analogsfor ¹²⁵ I-T₃ binding to in vitro translated rTR alpha.

FIG. VII-5 is a photograph of a gel which illustrates tissuedistribution of rTR alpha mRNA.

DEFINITIONS

In the present specification and claims, reference will be made tophrases and terms of art which are expressly defined for use herein asfollows:

As used herein, GR means glucocorticoid receptor. Disclosed DNA hGRcodes for glucocorticoid receptor GR.

As used herein, MR means mineralocorticoid receptor. Disclosed DNA hMRcodes for mineralocorticoid receptor MR.

As used herein, TR means thyroid receptor. Disclosed human DNA'sc-erb-A, hERBA 8.7 and hFA8, and rat rbeA12, all code for thyroidreceptor.

As used herein, hERR1 and hERR2 designate DNA's which code forestrogen-related receptor proteins.

As used herein, glucocorticoid hormones include cortisol, hydrocortisone(HC), and corticosterone (CS), and analogs thereof include dexamethasone(Dex), deoxycorticosterone (Doc), and triamcinolone acetonide.

As used herein, mineralocorticoids include aldosterone (Aldo), as wellas corticosterone (CS), and deoxycorticosterone (Doc).

As used herein, thyroid hormones include thyroxine (T4) andtriiodothyronine (T3).

As used herein, estrogens (or oestrogens) include estradiol-17 beta, andanalogs thereof include diethylstilbestrol.

As used herein, progestogens include progesterone (Prog), and analogsthereof include promegestone.

As used herein, androgens include dihydroxytestosterone, and analogsthereof include methyltrienolone.

As used herein, MTV means mammary tumor virus; MMTV means mouse mammarytumor virus.

As used herein, RSV means Rous sarcoma virus; SV means Simian virus.

As used herein, CAT means chloramphenicol acetyltransferase.

As used herein, COS means monkey kidney cells which express T antigen(Tag). See Gluzman, Cell, 23:175 (1981). COS cells are useful in thebioassay system of the present invention.

As used herein, CV-1 means mouse kidney cells from the cell linereferred to as "CV-1". CV-1 is the parental line of COS. Unlike COScells, which have been transformed to express SV40 T antigen (Tag), CV-1cells do not express T antigen. Like COS cells, CV-1 cells are useful inthe bioassay system and methods of the present invention.

As used herein, when it is said that a protein has "hormone-bindingproperties characteristic of a hormone receptor", it means that, if, inany standard assay for binding-affinity between a hormone from aspecies, or a synthetic analog thereof, and its cognate receptor(s) fromthat species, the affinity of the protein for the hormone or syntheticanalog is at least about 10% of the affinity of the hormone or analogand the cognate receptor(s) from that species.

As used herein, when it is said that the transcription-activatingproperty of a protein (X) is "characteristic" of that of a hormonereceptor (R), it means that, if, when tested in an assay such as the"cis-trans" receptor functionality bioassay system of the invention,(see Description of the Invention; also see Experimental Section II,especially FIG. II-1, plus the subsections labeled "Results" and"Experimental Procedures" which relate to use of the bioassay to showfunctional expression of hGR), the rate of expression from a gene (G)(whose transcription is activated by binding of: a receptor complexedwith hormone or hormone analog) is, when protein (X) is employed inplace of receptor (R), at least about 10% that shown when receptor (R)itself is used, as long as, in both the case of the "receptor" (R) and"protein (X)", the involved cells are bathed in the same concentrationof hormone or analog thereof.

As used herein, when it is said that a protein has "hormone-binding ortranscription-activating properties characteristic of a hormonereceptor", it is intended that the hormone receptor itself beencompassed within this definition.

As used herein, when it is said that the transcription of a gene (G) is"substantially activated by hormone (H), or hormone analog (aH)", itmeans that the transcription of gene (G) is induced by binding of: ahormone/receptor [(H) or (aH)/(R) or (r)] complex to chromatin nearwhere gene (G) is located. Under this definition (R) is meant todesignate "wild-type" or unaltered hormone receptors. The lower case (r)notation is meant to designate functional "engineered"or "modified"receptor proteins, or proteins encoded by mRNA variants of "wild-type"receptor genes.

As used herein, GRE's mean glucocorticoid response elements and TRE'smean thyroid receptor enhancer-like DNA sequences. GRE's areenhancer-like DNA sequences that confer glucocorticoid responsivenessvia interaction with the GR. See Payvar, et al., Cell, 35:381 (1983) andSchiedereit, et al., Nature, 304:749 (1983). TRE's are similar to GRE'sexcept that they confer thyroid hormone responsiveness via interactionwith TR.

As used herein, the terms "transcriptional control unit","transcriptional control element", "hormone responsive promoter/enhancerelement" and "DNA sequences which mediate transcriptional stimulation"mean the same thing, and are used interchangeably.

As used herein, in the phrase "operative hormone responsivepromoter/enhancer element functionally linked to an operative reportergene", the word "operative" means that the respective DNA sequences(represented by the terms "hormone responsive promoter/enhancer element"and "reporter gene") are operational, i.e., work for their intendedpurposes; the word "functionally" means that after the two segments arelinked, upon appropriate activation by a hormone-receptor complex, thereporter gene will be expressed as the result of the fact that the"hormone responsive promoter" was "turned on" or otherwise activated.

As used herein, the term "receptor-negative" means that no receptor isdetectable in the cell, or if it is, only a de minimus amount (i.e., abarely detectable amount) of receptor is present.

As used herein, a "mutant" of a DNA of the invention means a DNA of theinvention which has been genetically engineered to be different from the"wild-type" or unmodified sequence. Such genetic engineering can includethe insertion of new nucleotides into the wild-type sequences, deletionof nucleotides from the wild-type sequences, or a substitution ofnucleotides in the wild-type sequences.

Use of the term "substantial sequence homology" in the presentspecification and claims means it is intended that DNA or RNA sequenceswhich have de minimus sequence variations from the actual sequencesdisclosed and claimed herein are within the scope of the appendedclaims.

The amino acids which comprise the various amino acid sequencesappearing herein may be identified according to the followingthree-letter or one-letter abbreviations:

    ______________________________________                                                       Three-Letter                                                                             One-Letter                                          Amino Acid     Abbreviation                                                                             Abbreviation                                        ______________________________________                                        L-Alanine      Ala        A                                                   L-Arginine     Arg        R                                                   L-Asparagine   Asn        N                                                   L-Aspartic Acid                                                                              Asp        D                                                   L-Cysteine     Cys        C                                                   L-Glutamine    Gln        Q                                                   L-Glutamic Acid                                                                              Glu        E                                                   L-Glycine      Gly        G                                                   L-Histidine    His        H                                                   L-Isoleucine   Ile        I                                                   L-Leucine      Leu        L                                                   L-Lysine       Lys        K                                                   L-Methionine   Met        M                                                   L-Phenylalanine                                                                              Phe        F                                                   L-Proline      Pro        P                                                   L-Serine       Ser        S                                                   L-Threonine    Thr        T                                                   L-Tryptophan   Trp        W                                                   L-Tyrosine     Tyr        Y                                                   L-Valine       Val        V                                                   ______________________________________                                    

The nucleotides which comprise the various nucleotide sequencesappearing herein have their usual single-letter designations (A, G, T, Cor U) used routinely in the art.

In the textual portion of the present specification and claims,references to Greek letters are written as alpha, beta, etc. In theFigures the corresponding Greek letter symbols are sometimes used.

Expression plasmid pGEM3 is commercially available from Promega Biotec,2800 South Fish Hatchery Road, Madison, Wis. 53711.

DEPOSITS

Plasmids pRShGR-alpha, pRShMR, peA101, rbeA12 and GMCAT, all of whichare in E. coli HB101, plasmids pE4 and pHKA, both of which are in E.coli DH5, plus plasmids phH3, phERBA 8.7 and phFA 8, have been depositedat the American Type Culture Collection, Rockville, Md., U.S.A. (ATCC)under the terms of the Budapest Treaty on the International Recognitionof Deposits of Microorganisms for Purposes of Patent Procedure and theRegulations promulgated under this Treaty. Samples of the plasmids areand will be available to industrial property offices and other personslegally entitled to receive them under the terms of said Treaty andRegulations and otherwise in compliance with the patent laws andregulations of the United States of America and all other nations orinternational organizations in which this application, or an applicationclaiming priority of this application, is filed or in which any patentgranted on any such application is granted.

The ATCC Deposit Numbers for the ten deposits are as follows:

    ______________________________________                                               pRShGR-alpha    67200                                                         pRShMR          67201                                                         peA101          67244                                                         rbeA12          67281                                                         GMCAT           67282                                                         pE4             67309                                                         pHKA            67310                                                         phERBA 8.7      40374                                                         phFA 8          40372                                                         phH 3           40373                                                  ______________________________________                                    

SUMMARY OF THE INVENTION

In one aspect, the present invention comprises a double-stranded DNAsegment wherein the plus or sense strand of the segment contains asequence of triplets coding for the primary sequence of a protein whichhas hormone-binding and/or transcription-activating propertiescharacteristic of a hormone receptor protein selected from the groupconsisting of: a glucocorticoid receptor, a mineralocorticoid receptorand a thyroid hormone receptor. According to this aspect of theinvention, the double-stranded DNA segment is one which is capable ofbeing expressed into the receptor protein.

In another aspect, the invention comprises a single-stranded DNA, whichis the sense strand of a double-stranded DNA according to the invention,and an RNA made by transcription of a double-stranded DNA of theinvention.

In another aspect, the invention comprises plasmids which contain DNAillustrative of the DNA of the present invention. These plasmids havebeen deposited with the American Type Culture Collection for patentpurposes. The plasmids of the invention include plasmids selected fromthe group consisting of: pRShGR-alpha (ATCC #67200), pRShMR (ATCC#67201), peA101 (ATCC #67244), rbeA12 (ATCC #67281), GMCAT (ATCC#67282), pE4 (ATCC #67309), pHKA (ATCC #67310), phERBA 8.7 (ATCC#40374), phFA8 (ATCC #40372), and phH3 (ATCC #40373).

In still another aspect, the invention comprises a cell, preferably amammalian cell, transformed with a DNA of the invention. According tothis aspect of the invention, the transforming DNA is capable of beingexpressed in the cell, thereby increasing the amount of receptor,encoded by this DNA, in the cell.

Further the invention comprises cells, including yeast cells andbacterial cells such as those of E. coli and B. subtilis, transformedwith DNA's of the invention.

Still further the invention comprises novel receptors made by expressionof a DNA of the invention, or translation of an mRNA of the invention.According to this aspect of the invention, the receptors will be proteinproducts of "unmodified" DNA's and mRNA's of the invention, or will bemodified or genetically engineered protein products which, as a resultof engineered mutations in the receptor DNA sequences, will have one ormore differences in amino acid sequence from the corresponding naturallyoccurring "wild-type" or cognate receptor (i.e., the naturally occurringreceptor of known sequence with the greatest amino acid sequencehomology to the novel receptor). Preferably these receptors, whether"unmodified" or "engineered", will have at least about 10% of thehormone binding activity and/or at least about 10% of thetranscription-activating activity of the corresponding naturallyoccurring cognate receptor.

The invention also comprises a novel method for determining thefunctionality of hormone receptor proteins produced from the DNA's (ormRNA's) of the invention. The new method, which is referred to herein asthe "cis-trans" bioassay system, utilizes two plasmids: an "expression"plasmid and a "reporter" plasmid. According to the invention, theexpression plasmid can be any plasmid which contains and is capable ofexpressing a receptor DNA of the invention, or an engineered mutantthereof, in a suitable receptor-negative host cell. Also according tothe invention, the reporter plasmid can be any plasmid which contains anoperative hormone responsive promoter/enhancer element functionallylinked to an operative reporter gene.

In practicing the "cis-trans" bioassay of the invention, the expressionplasmid (containing a "receptor" DNA of the invention) and the reporterplasmid are cotransfected into suitable receptor-negative host cells.The transfected host cells are then cultured in the presence and absenceof a hormone, or analog thereof, which is able to activate the hormoneresponsive promoter/enhancer element of the reporter plasmid. Next thetransfected and cultured host cells are monitored for induction (i.e.,the presence) of the product of the reporter gene sequence. Finally,according to the invention, the expression and steroid binding-capacityof the receptor protein (coded for by the receptor DNA sequence on theexpression plasmid and produced in the transfected and cultured hostcells), is measured. (See FIG. II-2 for a schematic drawing of this"cis-trans" bioassay system.)

The "cis-trans" bioassay system is especially useful for determiningwhether a receptor DNA of the invention has been expressed in atransformed host cell; it is also useful in determining whether areceptor of the invention has at least about 10% of the binding activityof the corresponding naturally occurring cognate receptor, as well aswhether such a receptor has at least about 10% of thetranscription-activating activity of the corresponding naturallyoccurring cognate receptor.

Finally, it has been discovered, with the use of the DNA's of theinvention, that a necessary and sufficient condition, for activation oftranscription of a gene (G) whose transcription is activated by hormonescomplexed with receptors, is the presence of the hormone and itsreceptor in the same cell as (G). This discovery has enabled us toprovide improved compositions and methods for producing desired proteinsin genetically engineered cells.

Two of these methods are methods of the present invention. The first isa method for inducing transcription of a gene whose transcription isactivated by hormones complexed with receptors. The second is a methodfor genetically engineering a cell and then increasing and controllingproduction of a protein coded for by a gene whose transcription isactivated by hormones complexed with receptor proteins.

In discussing these two methods, a gene whose transcription is activatedby hormones complexed with receptor proteins will be referred to as gene(G). The hormone which activates gene (G) will be referred to as (H),and any of its analogs as (aH). The receptor protein will be referred toas (R), and functional modifications thereof as (r). Finally, the cellwhere gene (G) is located will be referred to as (C), and the proteincoded for by gene (G) will be referred to as (P).

According to the gene induction method of the invention, cell (C), whichcontains gene (G), is transformed by a DNA of the invention, which iscapable of being expressed in cell (C) and which codes for receptor (R)or a modified functional form (r) thereof; and the concentration ofhormone (H), or analog (aH), in cell (C) is increased to a level atleast sufficient to assure induction of expression of gene (G).

According to the method for engineering a cell and then producingprotein (P): gene (G), which codes for protein (P), is placed in cell(C) so that it is under the control of a transcriptional control elementto which hormone (H), when complexed with receptor (R), can bind,thereby inducing transcription of gene (G). Also according to thisprotein production method, both hormone (H) and receptor (R) are presentin cell (C). The presence of receptor (R) is assured by transformingcell (C) with a DNA of the invention which codes for receptor (R), or afunctional modified form (r) thereof. The presence of hormone (H), orits synthetic analog (aH) is assured by bathing transformed cell (C) ina bathing solution which contains hormone (H) or analog (aH). Then,according to the method, the transcription of gene (G) is controlled bycontrolling the concentration of (H) or (aH) in the bathing solutionused to bath transformed cell (C). By so controlling the transcriptionof gene (G), it is possible to control the production of protein (P) incell (C).

As those skilled in the art will appreciate, based on this teaching, itwill now be possible to engineer cells so that production of a protein(P), encoded by a gene (G) whose transcription is activated by ahormone/receptor complex, is controlled by simply assuring the presenceof hormone (H) and its receptor in cell (C) where gene (G) is located,and then controlling the concentration of hormone (H) or its analog thatis present in cell (C).

DESCRIPTION OF THE INVENTION

The present invention relates, in part, to DNA segments which code forproteins having the hormone-binding and/or transcription-activatingproperties characteristic of glucocorticoid, mineralocorticoid andthyroid hormone receptors. According to this aspect of the invention,these DNA segments are ones capable of being expressed, in suitable hostcells, thereby producing glucocorticoid, mineralocorticoid and thyroidhormone receptors or receptor-like proteins. The invention also relatesto mRNA's produced as the result of transcription of the sense stands ofthe DNA's of the invention.

The DNA's of the invention are exemplified by DNA's referred to hereinas: human glucocorticoid receptor DNA (hGR); human thyroid hormonereceptor DNA's (hTR: hTR alpha and hTR beta; hTR alpha is exemplified byhERBA 8.7 and hFA 8; hTR beta is exemplified by cellular or "c-erb-A");rat thyroid hormone receptor (rbeA12), which is the rat homolog of humanthyroid receptor alpha; human mineralocorticoid receptor (hMR); and newhuman steroid hormone receptors (hERR1 and hERR2). The sense strand cDNAnucleotide sequences, and the predicted primary protein sequences codedfor thereby, are shown in FIG. I-2 for hGR; in FIG. III-1 for humanc-erb-A and in FIG. III-7 for hERBA 8.7 and hFA 8; in FIG. IV-2 for hMR;in FIG. V-1 and V-2 for hERR1 and hERR2, respectively; and in FIG. VII-1for rat thyroid receptor rbeA12.

DNA's hGR, human c-erb-A, hERBA 8.7, hFA8, hMR, hERR1, and hERR2 arepreferred DNA's of the invention. Also preferred are the plasmids whichcarry these and other DNA's of the invention. Preferred plasmidsinclude: pRShGR-alpha, pRShMR, peA101, rbeA12, GMCAT, pE4, pHKA, phERBA8.7, phFA 8, and phH3.

In addition to pRShGR-alpha, preferred DNA's include modifications ofpRShGR-alpha which are designated herein as I9, I37, I102, I120, I204,I214, I262, I289, I305, I346, I384, I403, I408, I422, I428, I440, I448,I490, I515, I532, I550, and 1684, where "I" stands for "Insert", and thenumber following the "1" represents the DNA modification designation.Most preferred of the modified pRShGR DNA's are those which encodeproteins having at least about 10% of the transcription-activatingproperties characteristic of human glucocorticoid receptor; those DNA'sinclude I9, I37, I102, I120, I204, I214, I262, I289, I305, I346, I384,I403, I408, I422, I428, I440, I448, I490, I515, I532, I550, and I684.

Construction of pRShGR-alpha is detailed in the part of thespecification labeled "Experimental Section II". (See especiallysubsection II. F. (b), "Recombinant Plasmids".) Experimental Section IIalso details construction and properties of the pRShGR-alphamodifications referred to in the preceding paragraph.

With regard to the cDNA sequence for hGR shown in FIG. II-2, the two C'sat the 5'-end of the indicated sequence are part of the KpnI sitejoining the indicated segment to the 3' end of the segment whichincludes the RSV-LTR, and the T at the 3'-end of the indicated sequenceis a few bases 5' of the point where the indicated segment is joined tothe segment which includes the SV40 polyadenylation signal.

pRShMR was constructed in essentially the same manner as pRShGR-alphaand is essentially the same as pRShGR-alpha. Stated another way, withthe exception of minor modifications at the insertion sites, the hMRsegment shown in FIG. IV-2 replaces hGR, the sequence of which is shownin FIG. I-2. Like pRShGR-alpha, pRShMV contains the receptor protein DNAcoding sequence under the control of the promoter from Rous Sarcomavirus, plus the SV40 origin of replication. See Footnote 41 in theReference portion of Experimental Section IV; also see FIG. IV-4A;compare with FIG. II-1.

With regard to the hMR sequence shown in FIG. IV-2, the AG at the 5'-endof the segment is a few base pairs downstream of a HindIII site, wherebythe hMR segment is joined to the RSV-LTR-containing segment. The AA atthe 3'-end of the segment shown in FIG. IV-2 is a few bases upstream ofthe 5'-end of the segment which includes the SV40 polyadenylationsignal.

Plasmid peA101 carries the entire coding region of human thyroidreceptor c-erb-A. (The gene for c-erb-A has been localized to humanchromosome 3; the protein product encoded by this receptor gene is nowreferred to as hTR beta. See Experimental Section III; compare withExperimental Section VII.)

Plasmid peA101 was constructed by inserting the EcoRI fragment frompheA12 (see FIG. III-1) into the EcoRI site of expression vector pGEM3(Promega Biotec), in the correct orientation. For further detail on thisconstruction see Experimental Section III, subsection III. I., under theheading labeled: FIG. III-4 Methods.

In addition to the hTR receptor which has been localized to humanchromosome 3, we have discovered a second thyroid hormone receptor thatis distinct from the protein sequence predicted by plasmid peA101. Wehave now isolated and characterized this new and unexpected thyroidreceptor from both the rat and the human. In the rat this new thyroidhormone receptor is encoded by the DNA of plasmid rbeA12. (The DNA andpredicted primary protein sequence for rbeA12 is shown in FIG. VII-1) Inthe human, the new thyroid hormone receptor is encoded by plasmid clonehERBA 8.7, and its related clone hFA 8. (See FIG. III-7 for thesequences of hERBA 8.7) hERBA 8.7 and hFA8 are cDNA products from thesame gene. The DNA sequence of clone hFA 8 is identical with the DNAsequence for hERBA 8.7 (shown in FIG. III-7) with the followingexceptions. The hFA 8 sequence is shorter than the sequence shown inFIG. III-7. More specifically, nucleotides 1 a (G), through 514 an (A),of the ERBA 8.7 sequence are missing in the hFA 8 sequence. In additionthe hFA 8 sequence has a deletion which extends from the guanine (G) atbase pair position 1138 through the guanine (G) at base pair 1244. Thisdeletion eliminates amino acids 368, a (Glu), through 406, a (Gln) fromthe polypeptide encoded by the hFA 8 clone. As stated above, our initialthyroid receptor has been localized to chromosome 3. As we show inExperimental Section VII, the human gene for the new thyroid hormonereceptor has been localized to human chromosome 17. Rat thyroid receptorrbeA12 represents the rat homolog of the human gene product fromchromosome 17.

Because they are encoded by distinct genetic loci, the chromosome 17gene products are now classified as hTR alpha and the chromosome 3 geneproduct is classified as hTR beta. Although highly related, the alphaand beta gene products contain specific changes in their primary aminoacid sequence. Also, alpha and beta products display characteristicallydistinct patterns of expression.

The actions of thyroid hormones are widespread and dependent upon thesereceptors. Prior to our cloning of thyroid hormone receptor, thisreceptor had not been purified or biochemically characterized. Also, thescientific literature was entirely devoid of any evidence suggesting theexistence of multiple thyroid hormone receptor gene products. Theexistence of multiple receptors will be useful as the basis fordeveloping thyroid hormone analogs that selectively activate only oneclass of these receptors. This could have widespread clinical impact andthus represents an exciting and important discovery.

The initial thyroid hormone receptor we characterized was peA12, thereceptor now referred to as human thyroid receptor beta. We used thisbeta clone to screen, by molecular hybridization, a rat brain cDNAlibrary for related sequences. This led to the identification of plasmidrbeA12 and its subsequent identification as a novel thyroid hormonereceptor of the alpha class. Rat rbeA12 in turn was used as a molecularhybridization probe to clone the human homolog to the rbeA12 geneproduct. The human product is encoded by clones hERBA 8.7 and hFA 8.

Additional thyroid receptor cDNA's (rat thyroid receptor rbeA12, andhuman thyroid receptors hERBA 8.7 and hFA8 can be expressed by insertingtheir cDNA's, in the correct orientation, into expression vector pGEM3,as was done for c-erb-A.

Turning now to plasmid GMCAT, it is a reporter plasmid that contains theMTV LTR linked to the bacterial gene for chloramphenicolacetyltransferase (CAT). As a result of this linkage, use of pGMCATprovides an enzymatic assay for assessing transcriptional activity ofthe MTV promoter. Since the MTV promoter contains several glucocorticoidresponse elements (GRE's), reporter plasmid pGMCAT can be cotransfectedwith expression plasmids carrying glucocorticoid or mineralocorticoidreceptor DNA's, now known or later discovered into suitable host cells.(Such cotransfection is part of the receptor "cis-trans" functionalitybioassay system of the present invention. This aspect of the inventionis discussed more fully below.) Detection of CAT activity in theco-transfected host cells show that the polypeptides produced by thereceptor expression plasmids are functional, i.e., have transcriptionactivating properties characteristic of receptor proteins. PlasmidpGMCAT has been deposited with the ATCC for patent purposes; it has beenaccorded ATCC #67282. (See FIG. IV-5 for a schematic drawing of pGMCAT.)

Plasmid GHCAT is an example of another reporter plasmid which is usefulin the present invention. pGHCAT contains a portion of the growthhormone promoter functionally linked to the bacterial gene forchloramphenicol acetyltransferase (CAT). Because of this linkage, use ofpGHCAT provides an enzymatic assay for assessing transcriptionalactivity of the growth hormone (GH) promoter. Since the GH promotercontains a thyroid hormone response element (TRE), reporter plasmidpGHCAT can be cotransfected with expression plasmids carrying thyroidhormone receptor DNA's, now known or later discovered, into suitablehost cells. (Such cotransfection is also part of the "cis-trans"receptor functional bioassay system of the present invention. Thisaspect of the invention is discussed more fully below.) When pGHCAT isused with a TR expression plasmid (which for example could carry hTRalpha or hTR beta DNA) to cotransfect suitable host cells, detection ofCAT activity in the co-transfected host cells can be used to show thatthe polypeptides produced by the thyroid receptor (TR) expressionplasmids are functional, i.e., have transcription activating propertiescharacteristic of thyroid receptor proteins.

Plasmids pE4 and pHKA relate to cDNA's which encode an estrogen relatedreceptor referred to herein as hERR1. (See Experimental Section V,especially FIG. V-1.) Plasmid pE4 carries the cDNA segment referred toin FIG. V-1 as lambda hKE4; pHKA carries the segment referred to in thatsame figure as lambda hKA1. Both pE4 and phKA1 have been deposited withthe ATCC for patent purposes. The two plasmids can be joined as followsto produce a single plasmid (pGMERR1) which contains the entire codingsequence for estrogen related receptor hERR1.

The preferred procedure for joining the two cDNA clones pE4 and pHKAmakes use of a synthetic linker which is complementary at each end for aspecific restriction enzyme site present in each cDNA. Morespecifically, the inserts from lamba hKA1 and lambda hKE4 are cloned asEcoRI fragments into the plasmid vector pGM3 (Promega Biotec); we namedthem pGMKA and pGMKE, respectively. Next, pGMKA is cut by NarI andHindIII and the fragment encoding hERR1 is purified from agarose gel(fragment 1). pGMKE is cut by DraIII and HindIII and the fragmentencoding the 5' end region of hERR1 and the vector sequences is purifiedfrom agarose gel (fragment 2). Thirdly, two synthetic oligonucleotidescomplementary to each other are synthesized. The oligonucleotides are asfollows:

Oligo I: GTGCCTGGTGCGGTGGGAGGAAAACCAGAGTGTATGCTACAAGCAGCCGGCGGG;

Oligo II: CGCCCGCCGGCTGCTTGTAGCATACACTCTGGTTTTCCTCCCACCGCACCAGGCACTTT.

Finally, Fragment 1 and 2 and Oligo I and II are ligated to each otheraccording to standard methods known well to those skilled in the art,and then transformed into the bacterial stain DH5. The resultingcolonies are screened for the DNA construct referred to herein aspGMERR1. Plasmid PGMERR1 can be used to express hERR1. See FIG. I-6 fora schematic drawing of pGMERR1.

Plasmid phH3 relates to clone lambda hH3 which was isolated from a humanheart lambda gtll cDNA library using a nick-translated 700-bp EcoRI-SmaIfragment representing the 5' portion of lambda hKA1. (Clones lambda hKE4and lambda hKA1 were isolated from a human kidney lambda gt10 cDNAlibrary; see Experimental Section V., H., especially the subsectionslabeled as FIG. V-1 and FIG. V-2 Methods.) Clone lambda hH3 carries cDNAwhich codes for an estrogen related receptor referred to herein ashERR2. The cDNA from phH3 can be inserted into pGM3 to create pGMERR2, adrawing of which is also shown in FIG. I-6. The functional andstructural characteristics of receptor-like polypeptides hERR1 and hERR2are disclosed and discussed in Experimental Section V.

One of the added discoveries we have made employing the DNA's of theinvention is the remarkable sequence homology among the various hormonereceptors, within one species, and, for any particular receptor, amongspecies. (See for example, FIG. III-2 which compares thecarboxy-terminal portions of the v-erb-A oncogene product, the humanplacental c-erb-A polypeptide, and the human glucocorticoid and estrogenreceptors; FIG. III-6 which compares the steroid and thyroid hormonereceptors; FIG. IV-3 which compares the amino acid homology betweenmineralocorticoid receptor and glucocorticoid receptor; FIG. IV-8 whichshows the amino acid comparisons between hGR, hMR, and hPR structures;FIG. V-3 which compares the carboxy-terminal regions of hERR1, hERR2,the human estrogen and human glucocorticoid receptors; and FIG. V-5which shows the amino acid comparison between hERR1, hERR2, hER andhuman thyroid hormone receptor, hT₃ R beta.) As a result of thishomology the DNA's and RNA's the invention can be used to probe for andisolate a gene from virtually any species coding for a hormone receptorwhich activates transcription by binding to chromatin DNA aftercomplexing with hormone. By so using the DNA's and RNA's of theinvention, especially the preferred DNA's that have been deposited withthe ATCC for patent purposes, those skilled in the art, without undueexperimentation, can screen genomic libraries to find otherglucocorticoid, mineralocorticoid and thyroid hormone receptors whichfall within the scope of the present invention. This aspect of theinvention is illustrated by our discovery of estrogen-related receptorshERR1 and hERR2 (see Experimental Section V, especially subsection A.,Introduction, and subsection B., cDNA Clones for Receptor hERR1), andrat thyroid receptor and human thyroid receptors TR alpha (seeExperimental Section VII, especially subsection C., Isolation of aSecond Thyroid Receptor DNA, and FIG. VII-1.)

DNA's and sense strand RNA's of the invention can be employed, inconjunction with the induction and protein production methods of theinvention for example, to make large quantities of substantially purereceptor proteins. In addition, the substantially pure receptor proteinsthus produced can be employed, using well known techniques, indiagnostic assays to determine the presence of specific hormones invarious body fluids and tissue samples.

Further, the receptor proteins of the invention can be employed inscreening for receptor-agonists and receptor-antagonists by usingbinding assays such as the one discussed in Experimental Section III forbinding T₃ to the receptor encoded by peA101, or in the "cis-trans"receptor functionality bioassay of the invention, which will bediscussed below.

Finally, because the receptor proteins of the invention can be producedin substantially pure form they can be crystallized, and their structurecan be determined by x-ray diffraction techniques. As will be apparentto those skilled in the art, such determinations are very useful whenengineering "synthetic" or modified receptor protein analogs.

In addition to DNA's and RNA's, and the novel receptor proteins producedthereby, the present invention discloses three general methods: onerelates to a bioassay for determining the functionality of receptorproteins; the other two relate methods for inducing and controllingexpression of genes whose transcription is activated by ahormone-receptor complex bound to chromatin DNA. Each of the threegeneral methods will be discussed separately.

The new bioassay system for testing receptor functionality, which werefer to as the "cis-trans" bioassay system, utilizes two plasmids: an"expression" plasmid and a "reporter" plasmid. According to theinvention, the expression plasmid can be any plasmid capable ofexpressing a receptor DNA of the invention, or a mutant thereof, in asuitable receptor-negative host cell. Also according to the invention,the reporter plasmid can be any plasmid which contains an operativehormone responsive promoter/enhancer element, functionally linked to anoperative reporter gene. (See the Definitions section of thisSpecification for an explanation of the terms used herein.) The plasmidspGMCAT and pGHCAT are examples of reporter plasmids which contain anoperative hormone responsive promoter/enhancer element functionallylinked to an operative reporter gene, and can therefore be used in thereceptor functionality bioassay of the invention. In pGMCAT, theoperative hormone responsive promoter/enhancer element is the MTV LTR;in pGHCAT it is the functional portion of the growth hormone receptor.In both pGMCAT and GHCAT the operative reporter gene is the bacterialgene for chloramphenicol acetyltransferase (CAT).

In practicing the "cis-trans" receptor functionality bioassay of theinvention, the expression plasmid and the reporter plasmid arecotransfected into suitable receptor-negative host cells. Thetransfected host cells are then cultured in the presence and absence ofa hormone, or analog thereof, able to activate the hormone responsivepromoter/enhancer element of the reporter plasmid. Next the transfectedand cultured host cells are monitored for induction (i.e., the presence)of the product of the reporter gene sequence. Finally, according to theinvention, the expression and/or steroid binding-capacity of the hormonereceptor protein, or mutant thereof (coded for by the receptor DNAsequence on the expression plasmid and produced in the transfected andcultured host cells), is measured. (See FIG. II-2 for a schematicdrawing of this "cis-trans" bioassay system.)

When using the "cis-trans" receptor functionality bioassay system of theinvention to determine the functionality of glucocorticoid ormineralocorticoid receptors, in preferred forms, plasmids will carry aselectable marker such as the amp gene. In addition, in preferred formsthe reporter plasmids will have the MTV LTR or a functional portion ofthe growth hormone promoter as the hormone responsive promoter/enhancerelement. MTV LTV is preferred because it is known that glucocorticoidhormones stimulate the rate of transcription of MTV DNA by increasingthe efficiency of transcription initiation at a unique site within theMTV LTR. Moreover, glucocorticoid receptors bind specifically to DNAsequences mapped within the MTV LTR, and thus can confer glucocorticoidresponsiveness to a heterologous promoter. (See Experimental Section II,especially subsection C. (a), Assay System and Experimental Design.) Itis also known that mineralocorticoid receptor shows functional kinshipwith the glucocorticoid receptor, and that the DNA binding domain of hMRrecognizes the MTV LTR. (See Experimental Section IV, especiallysubsection E.: Expression and Hormone Binding, and subsection F.:Transcriptional Activation). Growth hormone promoter is preferredbecause its activation is responsive to binding by the thyroidhormone-receptor complex.

Preferred host cells for use with the "cis-trans" bioassay system of theinvention are COS cells and CV-1 cells. (See Experimental Section II,subsection C. (a) Assay System and Experimental Design, for use of thepreferred host cells in the bioassay system of the present invention.)COS-1 (referred to as COS) cells are mouse kidney cells that expressSV40 T antigen (Tag); CV-1 do not express SV40 Tag. CV-1 cell areconvenient because they lack any endogenous glucocorticoid ormineralocorticoid or other known steroid or thyroid hormone receptors.Thus, via gene transfer with appropriate expression vectors, it ispossible to convert these host cells from receptor negative to receptorpositive. The presence of Tag in the COS-1 derivative lines allows theintroduced expression plasmid to replicate and provides a relativeincrease in the amount of receptor produced during the assay period.

Expression plasmids containing the SV40 origin of replication (ori) canpropagate to high copy number in any host cell which expresses SV40 Tag.Thus our expression plasmids carrying the SV40 "ori" can replicate inCOS cells, but not in CV-1 cells. Although the increased expressionafforded by high copy number is desired, it is not critical to thedisclosed bioassay system. The use of any particular cell line as a"host" is also not critical. The expression vectors are so efficientthat, in our hands, the assay has worked in all the hosts we haveexamined. CV-1 cells are preferred only because they are particularlyconvenient for gene transfer studies and provide a sensitive andwell-described host cell system.

The "cis-trans" bioassay system is especially useful for determiningwhether a receptor DNA of the invention has been expressed in atransformed host cell; it is also useful in determining whether areceptor of the invention has at least about 10% of the binding activityof the corresponding naturally occurring cognate receptor, plus whethersuch a receptor has at least about 10% of the transcription-activatingactivity of the corresponding naturally occurring cognate receptor.

FIG. II-2 schematically illustrates use of the "cis-trans" receptorfunctionality bioassay system of the invention when used to determinethe functionality of receptor polypeptides coded for by hGR cDNA.Details of the bioassay, and its effectiveness as a quantifiablebioassay system to test receptor functionality, are disclosed anddiscussed in Experimental Section II. (See especially, subsection F.,Experimental Procedures, and subsection C., (b), Expression ofFunctional hGR.) As that experimental section shows, in addition to theCAT enzymatic assay, which can be used to show activation of the hormoneresponsive promoter/enhancer element, Western blot analysis of thetransfected host cells can be used to demonstrate synthesis of receptorpolypeptides which are indistinguishable with respect to mobility fromthe cognate receptors used as controls. Moreover, by using the"cis-trans" bioassay system of the invention, activation of thereceptors (produced in the transfected and cultured host cells) byspecific hormones can also be examined, as can their hormone-bindingcapabilities and characteristics. As Experimental Section IIdemonstrates, when this was done for hER, it was shown that the hGR ofthe invention is functional and binds with glucocorticoid hormones withthe same specificity and concentrations as does the cognate receptor.

Finally, as stated in the Summary section, by using the DNA's of theinvention we have discovered that a necessary and sufficient conditionfor activation of transcription of a gene (G), whose transcription isactivated by hormones complexed with receptors, is the presence of thehormone and its receptor in the cell (C) where (G) is located. (Themethod by which hormone (H) and receptor (R) effect gene G'stranscription is not fully understood. However, it is believed thatreceptor (R), when complexed with hormone (H), binds to specific DNAsites, referred to in the art as "transcriptional control elements" or"DNA sequences which mediate transcriptional stimulation", which arelocated on the chromatin near where gene (G) is located. This binding bythe hormone/receptor, or (H)/(R), complex seems to act in a way not yetunderstood, as a hormone dependent "switch" that "turns on", or in someother manner activates, the promoter for gene (G), and thus stimulatesthe transcription of the (G) gene.)

Our discovery has enabled us to provide improved compositions andmethods for producing desired proteins in genetically engineered cells.Two of these methods are methods of the present invention. The first isa method for inducing transcription of a gene whose transcription isactivated by hormones complexed with the receptors. The second is amethod for engineering a cell and then increasing and controllingproduction of a protein encoded by a gene whose transcription isactivated by hormones complexed with receptor proteins.

Again, in discussing these two methods, a gene whose transcription isactivated by hormones complexed with receptor proteins will be referredto as gene (G). The hormone which activates gene (G) will be referred toas (H), and any of its analogs as (aH). Receptor protein will bereferred to as (R), and functional modifications thereof as (r).Finally, the cell where gene (G) is located will be referred to as (C),and the protein coded for by gene (G) will be referred to as (P).

According to the gene induction method of the invention, cell (C), whichcontains gene (G), is transformed by a DNA of the invention, which iscapable of being expressed in cell (C) and which codes for receptor (R)or a modified functional form (r) thereof; and the concentration ofhormone (H), or analog (aH), in cell (C) is increased to a level atleast sufficient to assure induction of expression of gene (G).

As we show in Experimental Section II, when we used the induction methodof the invention, to our great surprise, the presence of (H) and (R) inthe cell (C) where gene (G) was located not only induced transcriptionof gene (G) but also increases production of protein (P) 500-1000 fold.This finding showed us that the induction method can be used to not onlyinduce transcription, but to increase and control it as well. Thisfinding also led us to develop our method for engineering a cell andthen controlling production proteins (P) coded for by genes (G) whosetranscription is activated by hormones complexed with receptors. Thismethod will be discussed more fully below.

Our induction method can also be used to increase and control productionof protein (P) by simply adjusting the concentration of hormone (H), oranalog (aH), available to cell (C). (As those skilled in the art willunderstand, by transforming cell (C) with a DNA of the invention, anadequate supply of (R) or (r) can be assured in cell (C) so that lack of(R) or (r) will no longer be a limiting factor in the transcription ofgene (G). This being the case, by simply increasing the amount of (H) or(aH) in the culture solution, it will be possible to increasetranscription of gene (G) and consequently the amount of protein (P)that is produced in (C) cells.)

The induction method of our invention can be used to induce expressionof any gene (G) that is under transcriptional control of atranscriptional control element activated by binding of a steroid orthyroid hormone receptor complexed with one of its hormones (H), oranalogs (aH) thereof, as long as: (1), a DNA is available which codesfor receptor (R), or a functional modified form (r) thereof which hasthe transcription-activating properties of (R); (2), cell (C) is a cellthat can be cultured; and (3), cell (C) can be transformed to expressthe (R)- or (r)-coding DNA needed to complex with hormone (H) or analog(aH).

Without undue experimentation those skilled in the art can use any ofthe deposited DNA's of the invention as probes to search genomiclibraries for (R)- or (r)-coding DNA's which are not now available. Oncefound, these DNA's, if expressible in the cell (C) where gene (G) islocated, can be used to transform (C) cells. Methods for transformingcultured cells are well known and can be used by those skilled in theart without undue experimentation. Also without undue experimentation,those skilled in the art can determine what the base level of (H) is incell (C), if any is present, as well as what the concentration of (H) or(aH) must be in order to induce and control transcription of gene (G),and thus production of protein (P). The requisite concentrations of (H)can be supplied to transformed (C) cells by adding (H) or (aH) to theculture solutions used to bath cultured (C) cells.

We have taught that a necessary and sufficient condition fortranscription of gene (G) is the presence of (H) or (aH) and (R) or (r)in the cell (C) where gene (G) is located, and that transcription ofgene (G), and therefore production of protein (P), can be induced andcontrolled by simply increasing the amount of (H) or (aH) in the culturesolutions used to bath transformed (C) cells. As those skilled in theart will appreciate, based on these teachings, it will now be possibleto engineer cells so that production of a protein (P), encoded by a gene(G) whose transcription is activated by a hormone/receptor complex, iscontrolled by simply assuring the presence of hormone (H) and itsreceptor in cell (C) where gene (G) is located, and then controlling theconcentration of hormone (H) or its analog that is present in cell (C).This concept is the basis for the cell engineering and proteinproduction method of our invention.

According to our engineered cell and protein production method: (1),cell (C) is engineered to contain gene (G) so that transcription of gene(G) is under the control of a transcriptional control element to whichan appropriate hormone/receptor, (H)/(R), complex can bind, therebyactivating transcription of gene (G); (2), cell (C), which now containsgene (G) under the control of a transcriptional control element, istransformed by a DNA of the invention, which is capable of beingexpressed in cell (C) and which codes for receptor (R) or a modifiedfunctional form (r) thereof; and (3), finally the concentration ofhormone (H), or analog thereof, in cell (C) is adjusted so that thetranscription of gene (G) is not only induced but effectively increasedand controlled by increasing controlling the amount of hormone (H) thatis available to cell (C) from the culture solution used to bathtransformed (C) cells. By so increasing and controlling gene (G)'stranscription, production of protein (P) is also increased andcontrolled.

As with the induction method, in our engineered cell and proteinproduction method, both hormone (H) and receptor (R) are present in cell(C). Again, as with the induction method, the presence of receptor (R),or a functional modified form (r) thereof is assured by transformingcell (C) with a (R)- or (r)-coding DNA of the present invention. Asstated above, methods for transforming cultured cells are well known andcan be used by those skilled in the art without undue experimentation.The presence of (H), or its analog (aH), is assured, and theconcentration of (H) or (aH) is controlled, by simply bathingtransformed (C) cells in bathing solutions which contain appropriateconcentrations of (H) or (aH). Appropriate concentration, i.e.,concentrations of (H) or (aH) needed to for cell (C) to produce a givenamount of protein (P) can be determined in a given situation by thoseskilled in the art, without undue experimentation.

Again, as those skilled in the art will understand, by transforming cell(C) with a DNA of the invention, an adequate supply of (R) or (r) can beassured in cell (C) so that lack of (R) or (r) will no longer be alimiting factor in the transcription of gene (G). This being the case,by simply increasing the amount of (H) or (aH) in the culture solution,it will be possible to increase the amount of protein (P) that isproduced in (C) cells.

As was true with our induction method, the engineered cell and proteinproduction method of our invention can be used to control expression ofany gene (G) that can be inserted into cell (C) so that it is undertranscriptional control of a transcriptional control element activatedby binding of a steroid or thyroid hormone receptor (R) complexed withone of its hormones (H)), or analogs thereof, as long as: (1), a DNA isavailable which codes to receptor (R), or a functional modified form (r)thereof which has the transcription-activating properties of (R); (2),cell (C) is a cell that can be cultured; and (3), the (R)- or (r)-codingDNA is capable of being expressed in cell (C) where gene (G) is located.

Again, without undue experimentation, those skilled in the art can useany of the deposited DNA's of the invention as probes to search genomiclibraries for (R)- or (r)-coding DNA sequences not now available. Oncefound, these DNA's, if expressible in cell (C) where gene (G) islocated, can be used in the engineered protein production method of thepresent invention.

Also without undue experimentation, those skilled in the art candetermine what the base level of (H) is in cell (C), if any is present,as well as what the concentration of (H) or (aH) must be in order toinduce and control transcription of gene (G), and thus production ofprotein (P). The requisite concentration of (H) needed to assure theproduction of a desired amount of protein (P) can be supplied totransformed (C) cells by adding (H) or (aH) to the culture solutionsused to bath cultured (C) cells.

Various aspects of the present invention are further explained andexemplified in the seven experimental sections which follow.Experimental Section I relates to human glucocorticoid receptor. Morespecifically, that section discloses the primary structure of hGR cDNA,as well its expression into a polypeptide which is functionallyindistinguishable from previously disclosed hGR proteins. ExperimentalSection II relates to functional domains of hGR. As that sectiondiscloses, GR contains at least four functional domains, two of whichwere expected and correspond to the predicted DNA-and steroid-bindingdomains, and two of which were not expected, and have potent effects ontranscription. Experimental Section III relates to thyroid hormonereceptor c-erb-A. As that section discloses, c-erb-A encodes a thyroidhormone receptor we now refer to as hTR alpha. Taken in conjunction withour unexpected discovery of a second thyroid hormone receptor (seeExperimental Section VII), the data disclosed about c-erb in Section IIIwill be extremely useful in gaining further knowledge about thyroidreceptor proteins. Experimental Section IV relates to humanmineralocorticoid receptor, which we show has a structural andfunctional similarity to glucocorticoid receptor. Experimental Section Vdiscloses a new and unexpected class of steroid hormone receptors werefer to as hERR1 and hERR2. These receptors provide the first evidencefor the existence of a novel steroid hormone system. In conjunction withour disclosure of a new "cis-trans" bioassay system, the newestrogen-related hERR1 and hERR2 receptors will provide the basis fordevelopment of an assay system that will systematically lead to theidentification of novel hormones. The identification of such novelsystems is likely to have widespread physiologic and clinicalsignificance. In Experimental Section VI we disclose some of our datarelating to the sites in a rat thyroid receptor c-erb-A oligonucleotidewhich we found were necessary for T₃ regulation. Such knowledge, takenin conjunction with Experimental III and our disclosure in ExperimentalSection VII of a new and unexpected thyroid hormone receptor that islinked to human chromosome 17, will be useful in characterizing thethyroid receptor proteins.

Without further elaboration, it is believed that one of ordinary skillin the art, can, using the preceding description, and the followingExperimental sections, utilize the present invention to its fullestextent. The material disclosed in the experimental sections, unlessotherwise indicated, is disclosed for illustrative purposes andtherefore should not be construed as being limitive in any way of theappended claims.

EXPERIMENTAL SECTION I Primary Structure and Expression of a FunctionalHuman Glucocorticoid Receptor cDNA I. A. Summary

Here we report the complete amino-acid sequence of the humanglucocorticoid receptor (hGR), deduced from human lymphoid andfibroblast cDNA clones. The sequence reveals various structural featuresof the receptor, including the major immunogenic domain and acysteine/arginine/lysine-rich region which may constitute a portion ofthe DNA-binding domain. We describe the use of the SP6 transcriptionvector system to generate analytical amounts of full-length protein, anddemonstrate that the cell-free translated protein is both immunoreactiveand possesses steroid-binding properties characteristic of the nativeglucocorticoid receptor. Weinberger, et al., (1985b) describes thehomology of the hGR sequence to that of the oncogene v-erb-A.

I. B. Introduction

The glucocorticoid receptor is widely distributed and expressed in manycultured cell lines, and the control of gene expression byglucocorticoids, therefore, has been widely studied as a model fortranscriptional regulation. A number of glucocorticoid-responsivetranscription units, including mouse mammary tumor virus (MMTV)(Ringold, et al., 1975; Parks, et al., 1974), mouse and humanmetallothionein (Hager, et al., 1981; Karin, et al., 1980), ratalpha_(2M) -globulin (Kurtz, et al., 1977) and rat and human growthhormone (Spindler, et al., 1982; Evans, et al., 1982; Robins, et al.,1982) genes have been identified. DNA sequences mediatingtranscriptional stimulation of several of these genes have beenlocalized. For MMTV, these sequences are discrete genomic regionsupstream of the transcriptional start site which appear to exert theiractions independently of orientation and position (Chandler, et al.,1983; Ostrowski, et al., 1984). The steroid/receptor complex appears tobind to these regulatory sequences and a purified receptor has been usedto define the specific binding sites (Govinda, et al., 1982;Scheidereit, et al., 1983; Pfahl, 1982; Payvar, et al., 1983). Based onthe footprinting analyses of several responsive genes, a consensus DNAbinding sequence sharing the core sequence 5' TGT/CTCT 3' has beenproposed (Karin, et al., 1984).

The ability of the glucocorticoid-responsive element (GRE) to alter itsposition and orientation yet still maintain promoter inducibilitysuggests that it resembles the class of cis-acting regulatory sequencestermed enhancers (Chandler, et al., 1983). First discovered in virusesand subsequently in cellular genes, these sequences are necessary forefficient transcription in vivo (Laimonis, et al., 1982; Benoist, etal., 1981; Baerji, et al., 1983). It has been suggested that enhancersare recognized by trans-acting factors that mediate regulatory effectsby tissue-specific transcriptional control. Although the enhancerfactors have not been well characterized, the glucocorticoid receptormay serve as a paradigm for these putative gene activator proteins.

The availability of radiolabeled high-affinity glucocorticoid analoguessuch as dexamethasone and triamcinolone acetonide has led to thedevelopment of purification strategies resulting in the isolation ofnearly pure rat and human receptors (Simons, et al., 1981; Gehring, etal., 1983). Although the receptor migrates as a dimer in sucrosegradients, analysis on denaturing SDS-polyacrylamide gels detects asingle polypeptide of relative molecular mass (M_(t))˜94,000 (94K)(Westpahl, et al., 1982; Wrange, et al., 1979). The native polypeptidecontains intrinsic specificity for steroid binding and DNA sequencerecognition. By using as probes monoclonal and polyclonal antibodiesraised against the purified rat and human receptors (Okret, et al.,1981; Harmon, et al., 1984; Gametchu, et al., 1983), it has beenpossible to identify a major immunogenic region in the receptor residingon a portion of the molecule that is distinct from the steroid- andDNA-binding regions (Carstedt-Duke, et al., 1982; Wrange, et al., 1984;Dellweg, et al., 1982). To gain further information about the structureof this molecule and to begin an analysis of the molecular mechanisms bywhich it regulates gene transcription, we set out to clone receptor cDNAsequences. By using receptor-specific antibodies as probes, we andothers have isolated clones containing human or rat glucocorticoidreceptor cDNA inserts (Weinberger, et al., 1985a; Miesfeld, et al.,1984).

I. C. Results (A) Glucocorticoid receptor cDNA

A library of cDNA clones was constructed in the phage expression vectorlambda gtll using poly (A)⁺ RNA from the human lymphoid cell line IM-9as template, as described previously (Weinberger, et al., 1985a). Thislibrary was initially screened with a rabbit polyclonal antiserum to thepurified glucocorticoid receptor, resulting in the isolation of severalimmunopositive candidate clones from ˜2.5×10⁵ plaques. Thebeta-glactosidase fusion proteins generated from these clones were usedto affinity-purify receptor epitope-specific antibody, which wassubsequently recovered and identified by binding to protein blots ofcellular extracts. Three clones containing inserts expressing antigenicdeterminants of the human glucocorticoid receptor were isolated. Theinserts of these clones, although of different sizes, cross-hybridized,indicating that they contained a common sequence which presumablydelimits the major immunogenic domain of the receptor. Together, theseclones spanned 1.4 kilobase pairs (kbp) but were clearly not long enoughto code for the entire receptor, which was estimated to require ˜2,500nucleotides to encode a polypeptide of M_(t) 94K.

To isolate additional cDNA clones we again screened the original libraryand also examined a second library (given by H. Okayama) prepared withpoly(A)⁺ RNA from human fibroblasts in the vector described by Okayamaand Berg (1983). Using one of the immunopositive cDNA inserts (hGR1.2)as probe, 12 clones were isolated that, together, covered more than 4.0kbp. The nucleotide sequences of these clones were determined by theprocedure of Maxam and Gilbert (1977) according to the strategyindicated in FIG. I-1a. RNA blot analysis indicated that a cDNA insertof 5-7 kilobases (kb) would be necessary to obtain a full-length cloneand sequence analysis indicated that the overlapping clones OB7 andhGR5.16 spanned an open reading frame of 720 amino acids, not largeenough to encode the complete receptor. Therefore, a second humanfibroblast cDNA library of ˜2×10⁶ transformants was screened, yielding aclone (OB10) containing a large insert that extended 150 base pairs (bp)upstream of the putative translation initiation site (see FIG. I-1).Sequence analysis predicts two protein forms, termed alpha and beta,which diverge at amino acid 727 and contain additional distinct openreading frames of 50 and 15 amino acids, respectively, at their carboxytermini (see FIG. I-1b). The alpha form, represented by clone OB7, isthe predominant form of glucocorticoid receptor because eight cDNAclones isolated from various libraries contain this sequence.

(b) cDNA and protein sequences

FIG. I-2 shows the 4,800 -nucleotide sequence encoding the human alphaglucocorticoid receptor, determined using clones hGR1.2, hGR5.16, OB7and OB10. The translation initiation site was assigned to the methioninecodon corresponding to nucleotides 133-135 because this is the first ATGtriplet that appears downstream from the in-frame terminator TGA(nucleotides 121-123). However, in the absence of amino-terminal peptidesequence information, unequivocal determination of the initiation siteis not yet possible. The codon specifying the lysine at position 777 isfollowed by the translation termination codon TGA. The remainder of thecoding sequence is covered by multiple overlapping clones, with OB7containing a 4.3-kb insert that continues to the poly(A) addition siteand OB10 containing the putative initiator methionine. The 3' regions ofclones OB7 and OB10 diverge at nucleotide 2,314, as shown by bothrestriction endonuclease and DNA sequence analysis. At this junction,the alpha-receptor continues with a unique sequence encoding anadditional 50 amino acids whereas the beta-receptor continues for only15 additional amino acids (FIG. I-3). The 3'-untranslated region of OB7is 2,325 nucleotides long, while that of OB10 is 1,433 nucleotides.There is no significant homology between these two regions, as indicatedby direct sequence comparison (FIGS. I-2 and I-3) or by hybridizationanalysis under stringent conditions (data not shown).

In addition, we have isolated from a human primary fibroblast libraryanother cDNA clone, OB12 (data not shown), which contains sequencesidentical to OB7 but uses the polyadenylation signal at nucleotide 3,101(FIGS. I-1b and I-2), giving rise to a shorter 3'-untranslated region.Use of probes specific for the 3'-untranslated region of OB7 to screen ahuman placenta cDNA library reveals that most clones terminate at thefirst poly(A) site in OB7. Thus, messenger RNA variation is the apparentconsequence of both alternative polyadenylation and alternative RNAsplicing (see below). The fact that the human fibroblast librarycontained both cDNA's suggests that both receptor forms may be presentin the same cell.

(c) Analysis of alpha- and beta-receptor protein

Sequence analysis reveals that the alpha and beta forms of the humanglucocorticoid receptor are 777 and 742 residues long, respectively; thetwo forms are identical up to residue 727, after which they diverge. Toexamine the receptor levels in vivo, cytoplasmic extracts from severalhuman and mouse cell lines were probed by immunoblot analysis with apolyclonal antibody directed against the human glucocorticoid receptor(Harmon, 1984). Alpha- and beta-receptor cDNA's were inserted into theSP6 transcription vector to create synthetic mRNA for in vitrotranslation (FIG. I-4a). The RNA's were separately added to a rabbitreticulocyte lysate system and the unlabeled products analyzed bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). The two RNA's programthe synthesis of distinct translation products whose migrationdifferences are consistent with the predicted polypeptide lengths of thetwo forms (FIG. I-4b, lanes 2, 3). Cytoplasmic extracts from untreatedIM-9 cells and IM-9 cells treated with 1 microM triamcinolone acetonideserve as markers (FIG. I-4b, lanes 4,5) for the 94K receptor (the 79Kform represents a putative receptor degradation product) (Wrange, etal., 1984). Note that after steroid treatment, the intensity of the 94Kband is reduced, corresponding to tighter receptor/chromatin bindingand, therefore, receptor translocation to the nucleus. The alpha formco-migrates with the 94K band of the negative receptor while the betaform migrates more rapidly (see FIG. I-4b, compare lanes 2,3 with lanes4,5). A comparison of cytoplasmic extracts from various human and mousecell lines reveals the presence of only the alpha-receptor (see FIG.14b, lanes 6-9). Interestingly, the mouse ADR6 lymphoma cell line(Danielsen, et al., 1984), selected for resistance to steroid-inducedlysis, contains no steroid-binding activity and shows no immunoreactivereceptor (see FIG. I-4b, lane 7). Therefore, based on characterizationof multiple receptor cDNA clones and receptor protein by immunoblotanalysis, we conclude that the predominant physiological form of theglucocorticoid receptor is the alpha (94K) species.

(D) Expression of hGR in vitro

To provide additional evidence that the cloned receptor is functional,we investigated the possibility that the in vitro-translated productsmight be able to selectively bind corticosteroids. Accordingly, therabbit reticulocyte lysate was incubated with the radiolabeled syntheticglucocorticoid analogue ³ H-triamcinolone acetonide (³ H-TA) before orafter addition of in vitro-synthesized alpha or beta hGR RNA. As shownin FIG. I-5, those lysates programmed with alpha-hGR RNA acquiredselective steroid-binding capacity; unexpectedly, the beta-receptorsynthesized in vitro failed to bind competable ³ H-TA. The invitro-synthesized alpha-hGR bound radiolabeled steroid which could becompeted with by addition of excess unlabeled cortisol or dexamethasone;however, binding of ³ H-TA was not effectively competed with by additionof excess unlabeled oestrogen or testosterone. In contrast, excessprogesterone constituted an effective competitor, consistent with thepreviously reported anti-glucocorticoid activities of progesterone(Rousseau, et al., 1972). To confirm these results, the competitionexperiments were repeated with native glucocorticoid receptor preparedfrom extracts of human lymphoid cells. Both the in vitro-translatedreceptor and the natural in vivo receptor have nearly identicalproperties with regard to steroid binding and competition with excessunlabeled steroid analogue (see FIG. I-5).

(e) hGR sequences map to at least two genes

The human glucocorticoid receptor gene has been functionally mapped tochromosome 5. Analysis of somatic cell hybrids constructed by fusingreceptor-deficient mouse T cells (EL4) with human receptor-containing Tcells (CEM-C7) indicated that segregants expressing the wild-type CEM-C7receptor maintained human chromosome 5 while dexamethasone-resistantsegregants had lost this chromosome (Gehring, et al., 1985).

To confirm the authenticity of our cDNA clones, we mapped receptor cDNAsequences using Chinese hamster/human somatic cell hybrids containingonly human chromosome 5 (HHW454). DNA's extracted from human placenta,HHW454 hybrid cells and Chinese hamster ovary (CHO) cells were digestedwith EcoRI or HindIII restriction endonucleases and separated on a 0.8%agarose gel. DNA fragments transferred to nitrocellulose were probedwith a portion of the receptor-coding region derived from nucleotides57-640 (See hGR1.2A in FIG. I-1). In addition to CHO-specific EcoRIbands of 6.8 and 17 kbp, DNA from the hybrid cell line also containshuman-specific bands of 3.0 and 5.0 kbp (see FIG. 6a, lanes 2, 3 ofHollenberg, et al., 1985). (The study disclosed herein as experimentalSection I was published as Hollenberg, et al., 1985. FIGS. 6 and 7appear in the paper but not in the present specification.) Unexpectedly,a DNA fragment of 9.5 kbp is found in total human DNA but not in thehybrid line (see Hollenberg, et al., 1985, FIG. 6a, lane 1). Similarly,HindIII digestion revealed a 7.5 bkp band that is not present in thechromosome 5 hybrid cell DNA (see Hollenberg, et al., 1985, FIG. 6a,lane 4). These results indicate that the receptor cDNA maps to humanchromosome 5, but that there are additional receptor-related sequenceselsewhere in the genome. To map these sequences, we used a dual-laserfluorescence-activated cell sorter (FACS) to sort mitotic chromosomesuspensions stained with DIPI/chromomycin in conjunction with Hoechst33258 chromomycin; this technique allows separation of the 24 humanchromosome types into 22 fractions (Lebo, et al., 1984). After thechromosomes were sorted directly onto nitrocellulose, the chromosomalDNA was denatured and hybridized to the hGR cDNA probe. In addition toconfirming the chromosome 5 localization, additional sequences werefound on chromosome 16 (see Hollenberg, et al., 1985, FIG. 6b ). Toconfirm this localization, DNA's from mouse erythroleukaemia cells and amouse erythroleukaemia cell line containing human chromosome 16 (seeBode, et al., 1981) were digested with HindIII and probed with hGR cDNA(see Hollenberg, et al., 1985, FIG. 6c); as predicted, the only DNAfragment found in the hybrid and not in the control was the 7.5-kbp DNAfragment, thus establishing the chromosome 16 assignment (seeHollenberg, et al., FIG. 6c, lanes 1, 2).

Additional Southern blot analyses using the EcoRI-XbaI fragments fromOB7 and OB10 3'-untranslated regions revealed hybridization only tochromosome 5 (data not shown). We conclude that both the alpha- andbeta-receptor cDNA's are probably encoded by a single gene on chromosome5 and suggest that the two cDNA forms are generated by alternativesplicing. In addition, we conclude that another gene residing on humanchromosome 16 contains homology to the glucocorticoid receptor gene, atleast between nucleotides 570 and 1,640. It is not clear whether thesesequences on chromosome 16 represent a related steroid receptor gene, aprocessed gene or pseudogene, or a gene that shares a common domain withthe gene for the glucocorticoid receptor. Genomic cloning and DNAsequencing may provide the answer.

To determine the size of the mRNA encoding the glucorcorticoid receptor,Northern blot hybridization (Bode, et al., 1981) experiments wereperformed using cytoplasmic mRNA isolated from a human fibroblast cellline, HT1080. Using the hGR1.2 coding sequence as probe, multiple mRNA'sof 5.6, 6.1 and 7.1 kb were detected. Treatment of these cells withglucocorticoids for 24 h leads to a 2-3-fold reduction in receptormRNA's, suggesting a potential negative feedback regulation.

I. D. Discussion

Structural analysis of the glucocorticoid receptor is a prerequisite forgaining insight into the mechanisms by which this regulatory moleculeexerts its effects on gene transcription. Here, we have presented theprimary sequence of the human glucocorticoid receptor deduced fromnucleotide sequence analysis of cDNA clones.

Isolation of hGR cDNA's has revealed the existence of multiple mRNA'sencoding at least two forms of the polypeptide. The predicted proteinsdiffer at their carboxy termini by the substitution of 50 amino acids inthe case of alpha-hGR and 15 amino acids in the case of beta-hGR. Thealpha glucocorticoid receptor is the major form identified in severalhuman cell lines and cDNA libraries. However, a recent report byNorthrop, et al. (1985) characterizes two forms of the receptor in mouselymphoid cells. The relationship of alpha- and beta-hGR to the mousedoublet species remains to be established. Also, the cellulardistribution and potential function of beta-hGR are unclear, although itis possible that variant receptors are used for tissue-specificfunctions. We are now generating antisera to synthetic peptides specificfor each human receptor form to elucidate their tissue-specificexpression.

Among the cDNA's selected using the immunopositive phage DNA inserthGR1.2A as a probe were those containing 3' ends similar to OB7, exceptthat polyadenylation was signaled earlier by the use of an AATAAA atnucleotide 3,101. These clones have been isolated from both humanfibroblast and placental libraries (data not shown). Alternative poly(A)site selection is a feature of many eukaryotic transcription units(Darnell, 1982). In some instances, selection of poly(A) sites specifiesparticular polypeptide products (Amara, et al., 1982; Rosenfeld, et al.,1983; Alt, et al., 1980; Schwarzbauer, et al., 1983) while in othercases, alternative poly(A) site selection produces no change in theprimary structure of the polypeptide (Setzer, et al., 1982) while inother cases, alternative poly(A) site selection produces no change inthe primary structure of the polypeptide (Setzer, et al., 1982). Theselection of poly(A) sites during receptor transcription may (1) alterthe stability of the mRNA in a particular tissue, (2) lead to splicingchanges, or (3) be random, with no physiological consequence.

The in vitro translation studies described here provide direct evidencethat the cloned molecule encodes the complete glucocorticoid receptor.First, the in vitro-translated product is identical in size to thenative glucocorticoid receptor and is immunologically reactive withreceptor-specific antiserum. Second, the in vitro-translated proteinacts functionally as a glucocorticoid receptor in that it is capable ofselectively binding the synthetic glucocorticoid triamcinoloneacetonide. This binding is specifically competed with byglucocorticoids, glucocorticoid analogues and progesterone but is notcompeted with by the sex steroids testosterone and oestrogen. In thisrespect, the in vitro-translated receptor behaves identically to the invivo receptor from human lymphoid cells, providing the first evidence ofa function for the cloned molecule. The acquisition of steroid-bindingproperties does not appear to require any specific modifications or, ifit does, these modifications can occur in the in vitro translation mix.

The results presented here provide the information necessary forstudying the molecular interactions of a eukaryotic transcriptionalregulatory protein with its target genes. These structural studiesprovide a basis from which the glucocorticoid receptor, its gene, andits RNA products can be analyzed. Furthermore, the ability to expressreceptor in vitro provides a novel means by which the consequence ofspecific in vitro mutagenesis can be rapidly tested. Finally, theisolation of genes responsive to glucocorticoids and specific regulatoryelements by both mutagenic and protein-binding studies suggests thatthis protein can serve as a very useful model for analysis of inducibleeukaryotic gene regulation.

I. E. Detailed Description of Figures Referred to in ExperimentalSection V FIG. I-1

Human glucocorticoid receptor cDNA sequencing strategy and schematicrepresentation of cDNA clones. a, the composite cDNA for the alphaglucocorticoid receptor is represented at the top, with noncoding(lines) and coding (stippled portion) sequences indicated. Common6-nucleotide restriction enzyme sites are shown. Overlapping cDNAinserts used to determine the sequence are shown: arrows beneath theregions sequenced show the direction and extent of sequencing. Thedashed line at the 3' end of OB10 indicates divergent sequence. Numbersrefer to nucleotide positions in OB10 relative to the 5'-mosttranscribed sequence. b, cDNA's encoding the alpha and beta forms of thereceptor (OB7 and OB10, respectively). The 5' end of OB7 (broken lines)is contributed by the OB10 clone. Protein-coding information isrepresented by wide bars; untranslated sequences are indicated by thinbars. Nucleotides and amino acids are numbered above and below thecoding sequence, respectively. Common DNA sequences extend to nucleotide2,313 (amino-acid residue 727), at which point the alpha- andbeta-receptor forms diverge, with the alpha cDNA's (OB12, OB7)continuing in an open reading frame for 150 nucleotides (50 amino acids)and the beta cDNA (OB10) continuing for 45 nucleotides (15 amino acids;see FIG. I-3). Hexanucleotide signals (AATAAA) just upstream of thepoly(A) in the clones are indicated, with the first hexanucleotide inOB7 serving as poly(A) in OB12.

FIG. I-1 METHODS

The inserts hGR1.2, hGR2.9 and hGR5.16 were isolated from a lambda gtllIM-9 lymphoid cell cDNA library as described previously (Weinberger, etal., 1985). Two clones were isolated from cDNA libraries constructed byH. Okayama in pcD (Okayama, et al., 1983) using poly(A)⁺ mRNA from GM637human fibroblasts (OB7) and primary human fibroblasts (OB10). Screeningwas performed with the hGR1.2-cDNA, radiolabeled by nick-translationwith ³² P-dCTP. Sequences were determined by the chemical cleavagemethod of Maxam and Gilbert (1977).

FIG. I-2

cDNA and predicted protein sequence of human glucocorticoid receptor.The complete alpha coding sequence and OB7 3'-untranslated region areshown, with the deduced amino acids given above the long open readingframe. An upstream in-frame stop codon at nucleotides 121-123 andputative additional polyadenylation signals in OB7 are underlined.

FIG. I-3

Restriction map and nucleotide sequence of the 3' end of the humanglucocorticoid receptor beta cDNA. a, The common 6-nucleotiderestriction enzyme sites are shown for the 3'-untranslated region ofOB10. b, The cDNA sequence of the beta form (OB10) from nucleotide 2,281to 3,820 compared with the protein-coding information found in the3'-terminal coding portion of the alpha form (OB7). Amino acids encodedby each of the cDNA's are presented above the nucleotide sequences.Putative polyadenylation signals (AATAAA) in the 3'-untranslatedsequence of OBIO are underlined.

FIG. I-4

Immunoblot comparison of hGR translated in vitro with in vivo hGR fromcell extracts. a, The vectors constructed for in vitro transcription ofthe hGR cDNA sequence. The complete alpha (pGR107) and beta (pGR108)coding sequences were placed under the transcriptional control of theSP6 promoter in pGEM1. Vector sequences, noncoding cDNA sequences andcoding sequences are indicated by thin lines, thick bars and boxedregions, respectively. The poly(A) tract of .sup.˜ 60 nucleotides isindicated by A_(pi). Divergent coding sequences are indicated by stripedand stippled regions. b, Western blot analysis of in vitro translationproducts and cell extracts. Unlabeled translation products synthesizedin a rabbit reticulocyte lysate system with no added RNA (lane 1) orwith RNA synthesized from pGR108 (beta, lane 2) or pGR107 (alpha, lane3) were fractionated on a 7.5% SDS-polyacrylamide gel. Additional lanesare: cytoplasmic extracts from IM-9 (lane 4), IM-9 treated with 1 microMtriamcinolone acetonide (lane 5), HeLa (lane 6), ADR6.M1890.AD1 mouselymphoma (lane 7), S49 mouse lymphoma (lane 8) and EL4 lymphoma (lane9). Proteins were transferred to nitrocellulose and probed with anti-hGRantibody, followed by ¹²⁵ I-labeled Staphylococcus aureus protein A asdescribed previously (Weinberger, et al., 1985).

FIG. I-4 Methods

To construct an expression vector containing the entire alpha codingsequence shown in FIG. I-2, the 3' coding sequence of OB7 was fused toOB10 5' coding information. OB7 was partially digested with EcoRI,completely digested with XbaI, and the 1.20-kbp fragment wasgel-purified and ligated with EcoRI/XbaI-digested OB10 to produce theintermediate pOB107. The entire pOB107 cDNA sequence including the 5'poly(G) tract (11 nucleotides, nt) and 3' poly(A) tract (.sup.˜ 60 nt)was excised by partial PstI/complete BamHI digestion. The resultant3.5-kb fragment was gel-purified and inserted between the PstI and BamHIsites of pGEMl (Promega Biotec) to yield pGR107. Plasmid pGR108 wasdirectly constructed from pOB10 by partial PstI/complete BamHI digestionand insertion of the resulting cDNA insert into the corresponding sitesof pGEM1, Capped SP6 transcripts were synthesized from PvuII-linearizedpGR107 and pGR108, as described by Krieg and Melton (1984), withsimultaneous capping effected by reduction of the GTP concentration from400 to 100 microM and addition of m⁷ GppG (Pharmacia) to 500 microM.Transcripts were purified by P60 chromatography and translated withmicrococcal nuclease-treated rabbit reticulocyte lysate (Promega Biotec)in conditions suggested by the manufacturer. Preparation of IM-9 cytosolfrom steroid-treated cells was as described previously (Weinberger, etal., 1985). Size markers are phosphorylase B (97K), bovine serum albumin(66K) and ovalbumin (45K).

FIG. I-5

Steroid-binding of alpha-hGR translated in vitro. Binding to IM-9cytosol extract (stippled bars) and to reticulocyte lysate containingSP6-generated alpha-hGR RNA (GR107; open bars) are shown. Bars representbound ³ H-triamcinolone acetonide (TA) determined with a 100-fold excessof various steroid competitors; 100% competition was determined usingunlabeled TA as competitor. The values represent the mean of triplicatedeterminations, with error bars showing P<0.05. Steroid competitors aredexamethasone (Dex), cortisol (Cort), progesterone (Prog), testosterone(Test), and oestradiol (Oest).

FIG. I-5 Methods

Binding assays were performed in 100 microliters containing 10 mMTris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM sodium molybdate, 10dithiothreitol, 150 mM ³ H-TA (20 Ci mmol⁻¹ ; Amersham) and 10microliters translation mixture or 100 microgram fresh lM-9 cytosol.Unlabeled steroid competitor (15 microM) was added as indicated. After 2h at 0° C., samples were extracted twice for 5 min. each with 5microliter of 50% dextran-coated charcoal to remove unbound steroid, andcounted. Uncompeted and fully competed values for the alphaglucocorticoid receptor (GR107) were 490 and 290 c.p.m., respectively.Reticulocyte lysate translation mixtures without added transcript orprogrammed with beta-receptor SP6 RNA (GR108) contained no competable ³H-TA binding.

Additional Figures

The scientific study presented here as Experimental Section I waspublished in Nature, 318:635-641 (1985). The Nature publication containtwo Figures which are not included in this Experimental section. Thosefigures are: FIG. 6, Chromosome mapping analysis of hGR cDNA; FIG. 7,Northern blot analysis of hGR cDNA.

I. F. References Referred to in Experimental Section I

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EXPERIMENTAL SECTION II Functional Domains of the Human GlucocorticoidReceptor II. A. Summary

Human glucocorticoid receptor (hGR) produced in CV-1 cells viatransfection of an hGR expression vector functions as a necessary andsufficient factor for the transcriptional activation of the MTV-CATfusion gene. The magnitude of the induction (500-1000 fold) reveals thatthe hGR may act as a transcriptional "switch", converting a silentpromoter containing a glucocorticoid response element to an activatedstate. Stimulation of transcription of the MTV-CAT gene fusion by hGR isnot dependent on transcriptional factors that are limiting in CV-1cells. Characterization of 27 insertional mutants of the hGR allowed thelocation of at least four functional domains, two of which correspond tothe predicted DNA- and steroid-binding domains. The other two domainsare referred to as tau for their potent effects on transcription. Thisraises the possibility that other regions in the receptor are necessaryfor full transcriptional activation but are not specifically involved insteroid or DNA binding.

II. B. Introduction

The primary structure of two classes of steroid hormone receptors havebeen elucidated by cloning and sequencing of their cDNA. As ExperimentalSection I discloses, identification of cDNA's encoding the humanglucocorticoid receptor (hGR) revealed two forms of the protein, of 777(alpha) and 742 (beta) amino acids, which differ at their carboxyltermini. (The Experimental Section I disclosure has been published asHollenberg, et al., 1985.) The human estrogen receptor is a somewhatsmaller protein of 595 amino acids (Greene, et al., 1986; Green, et al.,1986). Amino acid sequence comparisons revealed extensive regions ofhomology not only between the two classes of receptors but also with thev-erb-A oncogene product of avian erythroblastosis virus (Weinberger, etal., 1985; Greene, et al., 1986; Green, et al., 1986). This supports thesuggestion that steroid receptor genes and the c-erb-A proto-oncogeneare derived from a common primordial ancestral regulatory gene(Weinberger, et al., 1985).

On the basis of the amino acid sequence of hGR deduced from the clonedcDNA (see Experimental Section I), the locations of functionally andimmunologically important regions of the protein have been proposed(Weinberger, et al., 1985). These include an immunological domainlocated in the amino-terminal half of the protein, a DNA-binding domainthat shows structural similarities with other DNA-binding proteins, andthe glucocorticoid-binding site localized near the carboxyl terminus ofthe molecule. However, the location of each domain is tentative, and nodomain involved in the activation of transcription itself has beenidentified. In this study, we sought to confirm the proposed sites ofthe functional domains within the hGR and to find other regions ofimportance by introducing amino acid alterations in the hGR protein. Wefirst developed a novel expression system in monkey kidney cells inwhich the synthesis of functional hGR is directed by the transcriptionof the cDNA under the control of the long terminal repeat (LTR) of theRous sarcoma virus (RSV). The functions of the synthesized receptor weremonitored by the induction of transcription of the mouse mammary tumorvirus (MTV) LTR as measured by chloramphenicol acetyltransferase (CAT)assays (Gorman, et al., 1982a) and steroid hormone binding. This newexpression system allowed us to investigate the effect of insertionalmutagenesis on the various functions of the receptor, which led us topropose a more detailed model of the domain structure of the hGR. Ourresults, based on the analysis of 27 insertional mutations, confirm thenotion that the glucocorticoid receptor is composed of discretefunctional domains (Weinberger, et al., 1985). In addition, theyidentify additional sequences outside the proposed DNA- andsteroid-binding domains, which, as stated above, we refer to as tau fortheir potent effects on transcription.

II. C. Results (a) Assay system and experimental design

The assay system and strategy used to study the expression of functionalhGR from the cloned cDNA (See Experimental Section I) is shown in FIG.II-1. In these experiments, a glucocorticoid-responsivepromoter/enhancer element linked to a reporter gene was introduced intoa receptor-negative cell. Thus, in principle, this construction shouldbe transcriptionally inactive. For our assay, we chose to use the MTVLTR fused to the sequence coding for chloramphenicol acetyltransferase(CAT) (EC2.3.1.28). It has been demonstrated previously thatglucocorticoid hormones stimulate the rate of transcription of MTV DNA(Ringold, et al., 1977) by increasing the efficiency of transcriptioninitiation at a unique site within the MTV LTR (Ucker, et al., 1983).Moreover, glucocorticoid receptors bind specifically to DNA sequencesmapped within the MTV LTR (Payvar, et al., 1983), which can conferglucocorticoid responsiveness to a heterologous promoter (Chandler, etal., 1983). Cotransfection of pMTVCAT (or pGMCAT) with a receptorexpression plasmid provides functional receptors that allow induction ofCAT activity upon treatment of the transfected cells with glucocorticoidhormone. In addition, biochemical studies such as steroid bindingactivity and Western blot analysis of the expressed receptors can beperformed simultaneously.

The expression vector linking the RSV LTR to full length hGR cDNA(pRShGR alpha) was designed to obtain high levels of expression in awide range of host cell types. The vector pRShGR alpha is a derivativeof pRSVCAT (Gorman, et al., 1982b) in which the coding sequence of theCAT gene was replaced by the hGR cDNA. The origin of replication of SV40was introduced into the vector to allow the recombinant plasmid topropagate to high copy numbers in COS-1 (referred to as COS) monkeykidney cells that express T antigen (Tag) (Gluzman, 1981). COS cells andparental cell line CV-1 offer the additional advantage of havingundetectable levels of glucocorticoid receptors (unpublished observationand FIGS. II-2 and II-3).

(b) Expression of functional hGR

The above assay was designed to overcome some of the major difficultiesencountered in studying the mechanisms of action of steroid hormonereceptors. These difficulties include low intracellular levels ofreceptor, possible heterogeneity of receptors, and lack of aquantifiable bioassay system to test receptor functions. Accordingly, wefirst looked at the relative amount of hGR that could be made by COScells transfected with pRShGR alpha. FIG. II-2 (right lane), a Westernblot analysis of transfected COS cells, demonstrates that COS cellssynthesized an hGR polypeptide of 94 kd that is indistinguishable withrespect to mobility from the hGR present in the IM9 cell line (leftlane). Moreover, the amount of hGR present in transiently transfectedCOS cells is greater than the level found in IM9 cells, which containbetween 100,000-200,000 receptors per cell (Harmon, et al., 1984). Thisexpression system not only provides us with cells carrying highintracellular levels of hGR, but eliminates the possibility of receptormicroheterogeneity, which could interfere in the functional study ofhGR.

To test the functional capability of the expressed hGR as a positivetranscriptional factor, we performed CAT assays with cell extractsobtained after cotransfection with pMTVCAT and pRShGR alpha.Transfection into both COS and the parental CV-1 cells was examined. Asexpected (Alwine, 1985), the presence of SV40 Tag in COS cells increasedbasal activity of the MTV LTR (data not shown). Thus, CV-1 cells, whichdo not express the SV40 Tag, were used to achieve maximal induction. Asshown in FIG. II-3, cotransfection of pMTVCAT with a control plasmiddoes not generate CAT activity in CV-1 cells. Similarly, cotransfectionof pMTVCAT and pRShGR alpha does not produce any CAT activity. However,treatment of the same cotransfected CV-1 cells with dexamethasone (DEX)turns on the transcription of the MTV-CAT fusion gene. The inductionfactor is very large (approximately 500-1000 fold) since basal levels ofCAT activity produced by pMTVCAT are barely detectible (often zero) inCV-1 cells. As a control experiment, we cotransfected the beta form ofthe hGR (see Experimental Section I). which was shown to be unable tobind steroids (see Table II-1). FIG. II-3 demonstrates that hGR beta isnot functional in our expression assay. The activation of transcriptionby hGR is also restricted to promoters containing aglucocorticoid-responsive element. When pMTVCAT was substituted forpMTIaCAT, a plasmid containing the regulatory region of the humanmetallothionein Ia gene, which is responsive to heavy metals but not toglucocorticoids, no induction of CAT activity was observed afterhormonal treatment of the transfected CV-1 (data not shown). Theseresults demonstrate that in cells the hGR acts as a necessary andsufficient factor that functions as a steroid-dependent transcriptionalswitch.

Based on this assay, the activation of the receptor by steroids could beexamined. As shown in FIG. II-4A, DEX exhibits an ED₅₀ value of 3 nM onhGR-induced CAT activity, which is in agreement with ED₅₀ valuesobserved for DEX (5 nM) in a variety of psychological actions.Specificity of hGR action was further tested by treating cotransfectedCV-1 cells with 100 nM testosterone, oestradiol, and progesterone. Thesesteroids failed to induce CAT activity with the exception ofprogesterone, which stimulated hGR function at a value of 1% of themaximal induction produced by DEX (data not shown). These resultsindicate that transfected CV-1 cells synthesize functional hGR thatinteracts with pharmacological ligands with the specificity andconcentrations of the natural receptor.

Studies on specific interaction between enhancer-containing moleculesand cellular components have shown that CV-1 cells contain limitingamounts of cellular factors required for the function of certain viralenhancers (Scholer and Gruss, 1984). In those cells, CAT activitygenerated by transfected pSV2CAT (Gorman, et al., 1982a) reaches aplateau at 0.3 pmol of plasmid per dish. Similarly, if the hGR interactswith limiting factors, we should be able to saturate the CAT activityinduced by transfection of increasing amounts of pRShGR alpha with aconstant quantity of pMTVCAT. In this experiment, 2 pmol (5 micrograms)of pMTVCAT DNA was used and increasing amounts of pRShGR alpha DNA wereadded, together with nonspecific carrier DNA, to yield a total of 30micrograms per dish. FIG. II-4B demonstrates that CAT activity could bedetected when as little as 0.03 pmol (100 ng) of pRShGR alpha per dishwas transfected and that no plateau in CAT activity was reached. Thesedata suggest that stimulation of transcription of the MTV-CAT fusiongene by hGR is not dependent on transcriptional factors that arelimiting in CV-1 cells.

(c) Mapping of functional domains in hGR

Understanding the mechanisms by which hGR regulates gene transcriptionfirst required the characterization of its functional domains. Based onlimited proteolysis studies of the glucocorticoid receptor(Carlsdedt-Duke, 1982; Dellweg, et al., 1982; Wrange, et al., 1984;Reichman, et al., 1984) and on the analysis of the primary structure ofthe hGR (see Experimental Section I), a model for the structure of thereceptor has been proposed (Weinberger, et al., 1985). This modelidentifies three ma3or domains--an immunological domain spanning fromamino acid 145 to 280, a DNA-binding domain extending from amino acids421 to 481, and a steroid-binding domain located near the carboxylterminus of the protein. To test this model, we generated 27site-specific insertional mutations in the glucocorticoid receptorcoding sequence via a linker-scanning approach. These geneticallyengineered mutants were then assayed for their ability to stimulate genetranscription and to bind steroid hormone.

To generate linker-insertion mutants of the hGR, the plasmid pRShGRalpha was first linearized by partial cleavage using a restrictionenzyme that cleaves DNA molecules with high frequency. The linear formof the plasmid was isolated and a BamHI linker was added to restore theopen reading frame encoding the hGR. The resulting mutants carry threeor four additional amino acids, which disrupt the wild-type sequence ofthe protein. Using this technique, we have generated a random series ofhGR mutants (FIG. II-5). The ability of these mutants to expressfull-length hGR was estimated by Western blot analysis. The amounts ofhGR produced were shown not to vary by more than 30%, and thus none ofthe mutants appear to destabilize the expressed protein.

The functional properties of each mutant are compared with that of thewild-type hGR in Table II-1. CAT activity induced by 12 out of 27 hGRmutants was comparable with wild-type level. Analysis of the 15 hGRmutants having a diminished or a complete loss of function, as assayedby induction of CAT activity, shows that they belong to four separategroups. A cluster of these mutants located between amino acids 120 and215 in the so-called immunogenic domain forms the first group. Althoughno specific function has been assigned to this region of the receptormolecule, three mutants (I120, I204, and I214) show decreased capacityto induce CAT activity. Those mutants retained their full ability tobind steroids.

Perhaps not surprisingly, the second group of defective mutants is foundin putative DNA-binding domain of the receptor. This domain iscysteine-rich and consists of two repeat units of about 25 amino acidseach, which could fold into a loop structure coordinated by a Zn²⁺ligand (Miller, et al., 1985). In mutant I422, the sequence motif Cys-X₂-Cys is changed to Cys-X₅ -Cys. The presence of the additional aminoacids completely abolishes receptor function. Mutant I440 bears asimilar insertion of four amino acids between the two other cysteinesinvolved in the formation of the first loop and also fails to induce anydetectable level of CAT activity. On the other hand, mutant I428 extendsthe length of the loop itself, from 13 to 17 amino acids. Althoughseverely diminished, induction of CAT activity by I428 is stillmeasurable. Steroid-binding capacity of all three mutants located in theDNA-binding domain was shown to be in the range of wild-type level. Thethird region affected by the mutations is located next to theDNA-binding domain. Mutants I488 and I490 show low levels of CATactivity but bind steroid efficiently. The fourth group covers the last200 amino acids of the receptor protein. Five mutants (I582, I589, I599,I626, and I696) show undetectable levels of CAT activity. This lack offunctional activity is correlated with their total incapacity to binddexamethasone. These results show that the steroid-binding regionencompasses a large portion of the protein, all clustered near the Cterminus. In contrast to the amino terminus of the molecule, this regionis extremely sensitive to changes in the primary structure of thereceptor.

                  TABLE II-I                                                      ______________________________________                                        Functional Properties of hGR Mutants                                                   Inserted    CAT        DEX                                           hGR      Amino Acids Activity (%)                                                                             Binding (%)                                   ______________________________________                                        alpha    --          100        100                                           I9       RIR         117        NT                                            I37      RIRA        95         NT                                            I102     GSV         130        NT                                            I120     RGSA        2          76                                            I204     RIR         3          125                                           I214     RGSA        2          79                                            I262     ADPR        97         NT                                            I289     RIR         125        NT                                            I305     ADPR        86         NT                                            I346     ADPR        19         107                                           I384     RIR         101        NT                                            I403     ADPR        114        NT                                            I408     ADPR        55         NT                                            I422     GSV         0          105                                           I428     RIRA        2          92                                            I440     ADPR        0          69                                            I488     GSV         15         96                                            I490     RIRA        10         115                                           I515     RIR         109        NT                                            I532     GSV         115        NT                                            I550     ADPR        5          19                                            I582     RIR         0           0                                            I589     GSV         0           0                                            I599     SDP         0           0                                            I626     ADPR        0           0                                            I684     RGSA        79         81                                            I696     RGSA        0           0                                            beta     C-terminal  0           0                                                     deletion                                                             ______________________________________                                    

CV-1 or COS cells were transfected with pRShGR alpha, pRShGR beta, or amutated hGR alpha and assayed for CAT activity and steroid-bindingcapacity. After transfection, CV-1 cells were cultured for 2 days in thepresence of 10 nM DEX before cell lysis and CAT assay; COS cells weremaintained in normal media. The two parameters are quantified aspercentage (%) of wild-type hGR activity. Amino acids inserted in hGRalpha are given in the one-letter code. NT means not tested. Differencesin amino acid composition between hGR alpha and hGR beta are representedin FIG. II-5 and in Experimental Section I.

II. D. Discussion

We have shown that hGR produced in CV-1 cells via transfection of an hGRexpression vector functions as a necessary and sufficient factor for thetranscriptional activation of the MTR-CAT fusion gene. The magnitude ofthe induction reveals the hGR may act as a transcriptional "switch",which can convert a silent promoter containing a glucocorticoid responseelement to an activated state. Unlike other transcriptional factors thatare constitutively active, stimulation of transcription by hGR istotally dependent upon the presence of glucocorticoid hormones (FIG.II-3 and II-4A). The production of an excessive quantity of the proteinwithin a cell is not sufficient to induce transcription of a regulatedgene. The mechanism by which hGR is activated by the hormone is poorlyunderstood but, in analogy with the cyclic AMP-binding protein, islikely to involve allosteric transitions within the protein (McKay andSteitz, 1981; Gages and Adhya, 1985).

We have observed that activation of transcription by hGR is notrestricted by factors present in limiting quantity in CV-1 cells (FIG.II-4B). These results suggest that the binding of hGR-steroid complex toa glucocorticoid-responsive enhancer is sufficient to increase theactivity of general transcriptional factors at nearby promoters. Similarproperties for several other transcriptional factors have been reported.For example, Adfl, a transcription factor that activates the proximalpromoter of the alcohol dehydrogenase (Adh) gene in D. melanogaster,binds the Adh template DNA in the absence of other protein factors andrequires only endogenous RNA polymerase Ii and a fraction containinganother general transcription factor to activate initiation of Adh RNAsynthesis (Heberlein, et al., 1985). In a different type of experimentusing the recombinant plasmid pSV2CAT, which contains SV40enhancer/promoter elements, Scholer and Gruss (1984) have shown arequirement of a cellular molecule(s) for the function ofenhancer-containing DNA. Their experiments indicated the presence of alimited amount of cellular factor(s) required for the activation of theCAT gene by the SV40 enhancer element. However, no exhaustion of generaltranscriptional factors was observed. These data suggest that themechanism of action of specific positive transcriptional factors islikely to involve alterations in chromatin structure induced by thefactor itself that would facilitate the activity of generaltranscription factors or the polymerase itself (Moreau, et al., 1981;Wasylyk, et al., 1983). It has been previously shown that glucocorticoidtreatment causes both reversible and persistent changes in chromatinstructure in DNA regions containing a segment of the MTV LTR (Zaret andYamamoto, 1984). The mechanism by which bound receptors protentiatepromoter activity remains to be completely elucidated. However, theavailability of a system that overexpresses hGR will facilitate thefuture studies on the molecular basis of transcription activation bypositive transcriptional factors.

The results of the characterization of the 27 insertional mutantssupports and extends our previous suggestion that the humanglucocorticoid receptor is composed of a series of functional domains.It is noteworthy that all mutants that affect steroid binding areclustered at the carboxyl terminus. In addition to suggesting that thisregion functions as a discrete domain encoding hormone specificity, theresults imply the possibility that the other domains identified withinthe receptor may serve discrete functions. Accordingly, the ability ofthe receptor to recognize and to interact with specific DNA sequencesappears to reside in the Cys-Lys-Arg-rich region, which is highlyconserved with the estrogen receptor and the oncogene product v-erb-a.It seems logical that mutations in these regions would diminish theability of the receptor molecule to activate transcription sinceactivation depends on both the ability of the ligand to induce anallosteric transformation and the ability of the transformed molecule torecognize and interact with the DNA. Based on the initial model ofsteroid receptor structure (Weinberger, et al., 1985), these wereexpected outcomes of a mutagenic characterization. The unexpectedoutcome, however, is the identification of at least two other regionsinfluencing transcriptional activity. This raises the intriguingpossibility that other domains are present in the receptor that arenecessary for transcriptional activation but are not specificallyinvolved in either steroid or DNA binding. Mutants I120, I204, and I214bind steroid with wild-type affinity but have diminished transcriptionalactivity. These mutants clearly demonstrate that this domain, which werefer to as tau₁, is functionally important and required to obtaincomplete activity of the hGR. Interestingly, nonfunctional truncatedmutants (i.e., 40 kd) found in several lines of glucocorticoid-resistantcells are retained in nuclei more efficiently than the wild-typereceptor, but fail to activate transcription (Yamamoto, et al., 1976;Andreasen and Gehring, 1981; Westphal, et al., 1984). The receptorfragment missing in these "increased nuclear transfer" (nt^(i)) mutantsis evidently the amino terminus of the protein since they retainhormone-binding capacity. We note that tau₁, coincides with the majorimmunogenic domain of the hGR (Weinberger, et al., 1985), indicatingthat it is probably on the external surface of the molecule.Speculations on how this domain can fulfill its functions includeself-interaction leading to receptor dimerization, possible interactionswith general transcriptional factors such as RNA polymerase II, and/ormodulation of DNA binding by exerting allosteric influence over theremainder of the activated receptor (Dellweg, et al., 1982). Tau₁ isenglobed by the amino terminus of the receptor, a region which is notheld in common with the smaller estrogen receptor. Perhaps the estrogenreceptor gains the equivalent function of this domain by interactingwith a second protein, or, alternatively, tau₁ may interact with otherresidues within the glucocorticoid receptor itself, as opposed tointeracting with other regulatory molecules. The other tau region (whichwe refer to as tau₂) that affects transcriptional activation is a regionthat is present in the estrogen receptor and the v-erb-A oncogene. Itslocation also suggests that it may act as a "hinge" region linking thesteroid- and DNA-binding domains. Thus, these mutants could block theallosteric transformation necessary for receptor activation.

A third region affected by amino acid insertions is located in theputative DNA-binding domain described by Weinberger, et al. (1985). Thisdomain is composed of two repeated units containing a Cys-Lys-Arg-richsequence and is the most intensively conserved when compared with thev-erb-a oncogene and the estrogen receptor (Weinberger, et al., 1985;Green, et al., 1986; Greene, et al., 1986). These repeated units werefirst observed in the factor TF-IIIa (Miller, et al., 1985) and havesince been found by sequence homology searches in a number of othernucleic acid-binding proteins (Berg, 1986). Based on experimental andtheoretical studies of the factor TFIIIa, Miller and colleagues (1985)proposed a novel mechanism by which proteins bind DNA molecules. Intheir model, each unit is folded into a "fingered" structure centered ona zinc ion. A finger could bind to a half-turn of DNA. Mutants I422 andI440 carry an amino acid insertion that disrupts the motif Cys-X₂ -Cyscentral to the finger model. These mutants are totally inactive withrespect to transcriptional activation but retain their ability to bindglucocorticoid hormone. Preliminary experiments revealed that thesemutants also fail to translocate to the nucleus after hormone treatmentand to bind DNA in vitro (S. Hollenberg, unpublished observations).These mutated receptors demonstrate the functional importance of thefinger motifs present in hGR. The third mutant located in this region,I428, has the finger extended by the addition of four amino acids.Transcriptional activity of I428 is greatly impaired (2% of wild-typelevel), but is still detectable. Thus, the loops are apparently moretolerant of change than the zinc-binding motif. The demonstration thatfinger-like domains are functionally important in hGR leads us topropose that steroid hormone receptors are metallo-proteins, which mayhave evolved from a primordial ancestral DNA-binding protein.

Together, these data suggest that the receptor is composed of a melangeof regulatory domains, which may have been pirated over evolutionarytime to condense into the primordial steroid hormone receptor, which inturn gave rise to the large family of hormone response genes present inthe mammalian genome. The transcriptional activity of this moleculedemonstrates its potential ability to act as a genetic switch, which isconsistent with the role of steroid hormones in activating a variety ofdevelopmental lineages and homeostatic functions. The design of themutations allows for the convenient generation of any desired set ofsmall or large deletional mutants and the ability to switch domainsbetween related molecules to study function. In conjunction with therapid and quantitative functional assays described in this section, itis now possible to direct specific questions as to the functional natureof the tau, DNA-, and steroid-binding domains.

II. E. Detailed Description of Figures Referred to in ExperimentalSection II FIG. II-1

Schematic Representation of the hGR functional assay

In this assay, an expression vector containing the hGR alpha cDNA or amutant derived from it is cotransfected into CV-1 or COS cells with aplasmid carrying the CAT gene under the control of the MTV LTR. Thecells are then cultured in the presence or absence of hormone. CV-1cells were used to monitor induction of CAT activity, and COS cells wereused to measure steroid-binding capacity and the expression of hGRprotein.

FIG. II-2

Expression of hGR protein

COS cells were mock-transfected (center lane) or transfected withplasmid pRShGR alpha (right lane) and analyzed 2 days later for thepresence of hGR protein. Crude cytoplasmic extracts were resolved bySDS-PAGE and analyzed by Western blot. Cytoplasmic extract from IM9cells was loaded on the same gel for comparison (left lane).

FIGURE II-3

Induction of CAT activity by hGR

Subconfluent CV-1 cells were cotransfected with either pRSVgal(control), pRShGR alpha, or pRShGR beta and the reporter plasmid pMTVCATand cultured for 2 days in the presence (+) or absence (-) of 10 nMdexamethasone. CAT assays were performed as indicated in ExperimentalProcedures (see Section II. F.). C, chloramphenicol; AC,3-acetylchloramphenicol.

FIG. II-4

Dose-response to DEX and titration of pRShGR alpha

(A) CV-1 cells cotransfected with pRShGR alpha and pMTVCAT (10micrograms each plasmid) were cultured in the presence of increasingconcentrations of dexamethasone. The apparent ED₅₀ value for DEX was 3nM. The levels of CAT activity were plotted as percentages of themaximal response observed in a particular experiment. No CAT activitywas detected in the absence of DEX.

(B) Titration. Increasing amounts of pRShGR alpha were cotransfectedinto subconfluent CV-1 cells with a constant amount of pMTVCAT (5micrograms). The plasmid pBR322 was used as carrier DNA to yield a totalof 30 micrograms DNA per plate. Cells were cultured for 2 days in thepresence of 10 nM DEX and CAT activity was measured and plotted as in(A).

FIG. II-5

Location of functional domains in hGR

The hGR is schematically represented with putative domains involved intranscription activation, tau₁, and tau₂, indicated by hatched areas.The DNA-binding domain is represented by a stippled box; thesteroid-binding domain, by a dotted box. The positions of BamHI linkerinsertions are indicated by triangles and circles. The numbers refer tothe amino acid position (see Experimental Section I) after which theinsertion occurs. Open symbols represent mutants capable of inducinghormone-dependent transcriptional activity at wild-type levels, asmeasured by CAT activity, and closed symbols indicate greatly diminishedor abolished function. The bar indicates the location of the divergentamino acids present in hGR beta, which is not functional.

II. F. Experimental Procedures

(a) Culture conditions

CV-1 and COS-1 cells were grown at 37° C. in Dulbecco's modified Eagle'smedium supplemented with 5% (v/v) fetal calf serum, 400 microg/mlampicillin, and 100 microg/ml streptomycin. Cells were passed every 3days and never allowed to reach confluency in order to obtain goodtransfection efficiency. All transfected cultures were maintained at 37°C. with 5% CO₂.

(b) Recombinant plasmids

Plasmid pRShGR alpha and pRShGR beta, which direct the synthesis of thetwo forms of hGR in CV-1 and COS cells, were constructed from three DNAfragments. The first fragment is derived from pRSVCAT (Gorman, et al.,1982b) and contains the RSV LTR, pBR322 sequences, and SV40polyadenylation site. To obtain this fragment, pRSVCAT was cut withHindIII and the ends repaired by treatment with the Klenow fragment ofDNA polymerase I. KpnI linkers were added to these ends by standardprocedures (Maniatis, et al., 1982), and the plasmid was subsequentlycut with HpaI, which removed the CAT coding sequence. The secondfragment contains the coding sequence of either hGR alpha or hGR beta.Plasmids pOB113 and pOB117 (S. M. Hollenberg, unpublished results),which contain the entire coding sequences of the alpha and beta form ofhGR, respectively, were cut with BamHI. The ends were repaired withKlenow, and the plasmids were cut with KpnI. Ligation of the first andsecond fragments created plasmid pRhGR alpha and pRhGR beta. The thirdfragment to be added consists of a PvuII-HindIII fragment containing theSV40 ORI obtained from the plasmid pSV2CAT (Gorman, et al., 1982a). Theends of this fragment were repaired by treatment with Klenow, and NdeIlinkers were added to them. This DNA fragment, containing the SV40 ORI,was then introduced into the single NdeI site present in pRhGR alpha andpRhGR beta. Finally, the single BamHI site present in these plasmids wasdestroyed and replaced with a XhoI site by insertion of a syntheticadaptor. The resulting plasmids are pRShGR alpha and pRShGR beta.Plasmids pMTVCAT and pMTIaCAT were gifts from S. Gould.

(c) Insertional mutagenesis

Insertion of amino acids disrupting the wild-type sequence of hGR alphawas performed by the following method. Full-length linear pRShGR alphaDNA was generated by partial digestion with restriction enzymes AluI,DpnI, and BstNI. In the case of DNA cut by BstNI, the ends were firstrepaired with Klenow. The DNA molecules were then fractionated byagarose gel electrophoresis, and the linear form of the plasmid wasextracted. BamHI linkers of 8- or 12-mer were added to restore theoriginal reading frame of the hGR amino acid sequence. Plasmids carryinga single BamHI linker in the coding region of hGR were sequenced (Maxamand Gilbert, 1980) to confirm the position of the linker and theintegrity of the hGR mutants.

(d) Cell transfection and CAT assay

The recombinant DNA constructs were introduced into CV-1 cells bycalcium phosphate coprecipitation (Wigler, et al., 1979) or into COScells by DEAE-dextran (Deans, et al., 1984). Each plasmid preparationused for transfection was purified using two consecutive CsCl-EtBrequilibrium gradients. After transfection with the CAT gene constructs,CV-1 cells were prepared for CAT assay as described by Gorman, et al.(1982a). The assays were performed with one third of the total cellularextract and an incubation time of 6 hr.

(e) Western blot analysis

Crude extracts from COS cells were prepared by lysis with a buffercontaining 10 mM Tris-CHl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% TritonX-100. Equal amounts of protein (100 micrograms) were resolved by 7.5%polyacrylamide gel electrophoresis, transferred to a nitrocellulosefilter, and probed with anti-hGR antibody GR884 (Harmon, et al., 1984),followed by ¹²⁵ I-labeled Staphylococcus aureus protein A. Filters wereair dried and exposed to film. The amount of receptor was quantitated byscanning the autoradiographs.

(f) Steroid binding assay

COS cells were lysed in hypotonic buffer containing 10 mM Tris-HCl (pH7.5), 10 mM NaCl, 1 mM EDTA, 5 micrograms/ml antipain, 5 micrograms/mlleupeptin, and 0.5 mM PMSF by Dounce homogenization and centrifuged 10min. at 15,000×g to yield the cytosolic fraction. Incubations wereperformed in hypotonic buffer adjusted to 100 mM NaCl and contained 100micrograms of protein from the cytosolic fraction and 2×10⁻⁸ [³ H]DEX(Amersham, 95 Ci/mmol) in total volume of 200 microliters. Nonspecificbinding was measured by the addition of 2×10⁻⁶ unlabeled DEX. Reactionswere carried out at 0° C. for 2 hr., followed by a 5 min. incubationwith 20 microliters of 50% dextran-coated charcoal (10:1 activatedcharcoal:dextran) and centrifugation at 15,000×g for 2 min. at 4° C.Supernatants were counted by liquid scintillation methods in a BeckmanLS-7800 liquid scintillation spectrophotometer. Each assay usually gave2500-3000 cpm [³ H]labeled steroid; unlabeled DEX competed for 70% ofthis binding.

II. G. References Referred to in Experimental Section II

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EXPERIMENTAL SECTION III The C-erb-A Gene Encodes a Thyroid HormoneReceptor III. A. Summary and Introduction

The human glucocorticoid receptor (hGR) complementary DNA has beensequenced (see Weinberger, et al., 1985b and Experimental Section I) andshown to be functionally active (see Experimental Section II).Interestingly, sequence analysis of the receptor showed it to be relatedto the product of the v-erb-A oncogene product of avian erythroblastosisvirus (AEV) (see Weinberger, et al., 1985). This led to the proposalthat the steroid receptors and the erb-A oncogene products share acommon primordial archetype and that the erb-A proto-oncogene productsmay also be proteins that bind to DNA enhancer elements. Recentcharacterization of the human estrogen (Green, et al., 1986 and Greene,et al., 1986), chicken progesterone (Jeltsch, et al., 1986 and Conneely,et al., 1986) and human aldosterone (J. Arriza, C. W. and R.M.E.,unpublished data) receptors further support these conclusions.

Accordingly, we started characterizing the human c-erb-A proto-oncogeneeven though its functional identification could not be ensured. Duringthe progress of these studies, advances were made in detailing thefunctional domains of the glucocorticoid receptor (See ExperimentalSection II) which indicated that the hGR hormone binding domain wasunusually large, encompassing the carboxy-terminal 300 amino acids. Thisentire region has distant but significant similarity to the carboxyterminus of v-erb-A which therefore focused our attention on classes ofmolecules that might exert transcriptional regulatory effects similar tothose of steroid hormones.

The molecular mechanism of thyroid hormone stimulation of geneexpression seems to be similar to that outlined for steroids (seeEberhardt, et al., 1980). Thyroid hormone is present in all chordatespecies examined and exerts profound effects on development anddifferentiation, such as metamorphosis in amphibians (see Eberhardt, etal., 1980). Like steroids, thyroid hormones may enter cells by passivediffusion and bind to high affinity nuclear receptors which in turnmediate a rapid and selective activation of gene expression (Tata, etal., 1966 and Oppenheimer, et al., 1972). Evidence favoring thishypothesis has come largely from studies of the induction of growthhormone and its messenger RNA in the somatotroph of the rat anteriorpituitary and in a number of related rat somatotrophic cell lines(Samuels, et al., 1973 and Tsai, et al., 1974). Thyroid hormones rapidlyincrease the transcription of the growth hormone gene in these cells(Martial, et al., 1977 and Evans, et al., 1982). The increase intranscription is accompanied by increased levels of nuclear thyroidhormone-receptor complexes, is time- and concentration-dependent, and isindependent of protein synthesis (see Samuels, et al., 1976; Spindler,et al., 1982; and Yaffe, et al., 1984).

The similarity of steroid and thyroid hormone actions led us to examinethe possibility that the erb-A protein may itself be the thyroid hormonereceptor. We first isolated and characterized a cDNA for human c-erb-A.The sequence predicts a 456 amino acid polypeptide containing acysteine/lysine/arginine-rich region similar to the putative DNA-bindingdomain of steroid hormone receptors and a carboxy-terminal regiondistantly related to the steroid binding domain. Using the functionalassay developed for analyzing the hormone binding properties of clonedsteroid receptors (see Experimental Section I), we demonstrated that thetranslation product from the human c-erb-A cDNA possesses intrinsicthyroid hormone binding activity, characteristic of the native thyroidhormone receptor molecule.

III. B. Characterization of c-erb-A cDNA's

To isolate a human c-erb-A cDNA, a 500-base pair (bp) PstI DNA fragmentisolated from a region of the AEV genome containing only the v-erb-Agene (Vennstrom, et al., 1980) was used as a ³² P-labeled probe toscreen two human placenta cDNA libraries. Two overlapping lambda gt10cDNA clones were obtained by screening two independent libraries of.sup.˜ 10⁶ phage recombinants each. The restriction maps were deducedfor each of the cDNA clone EcoRI inserts from pUC8 subclones, pheA4 andpheA12 (FIG. III-1a). Nucleotide sequence analysis of these overlapping1.5 kilobase (kb) cDNA clones revealed that the composite sequencecontains a long open reading frame of 456 amino acids with a presumptiveinitiator methionine codon at nucleotide number 301 and a terminatorcodon at 1,669 in the sequence (FIG. III-1b). Seven codons upstream ofthe ATG is an in-frame terminator TAA providing support for theinitiator methionine, although another methionine found 26 codonsdownstream makes this assignment tentative. No consensus polyadenylationaddition signal (AATAAA, see Proudfoot, et al., 1976) is discernible inthe 27 nucleotides between the terminator and poly(A) tract in pheA12.

A predicted polypeptide of relative molecular mass 52,000 (M₁ =52K),encoded within the human c-erb-A cDNA translational open reading frame,shares 82% amino acid identity with the region downstream from the viralgag sequences (Debuire, et al., 1984) in AEV. No gaps in the amino acidcomparison were revealed. The human c-erb-A amino acid sequence ishomologous with the viral protein beginning at viral amino acid residue37 (FIG. III-2). The carboxy-terminus of c-erb-A differs from that ofv-erb-A in the following manner: amino acid sequence similarity of thetwo polypeptides terminates at residue 445 of c-erb-A and residue 380 ofv-erb-A (FIG. III-2).

Alignment of the human c-erb-A nucleic acid sequence with that ofv-erb-A shows that .sup.˜ 74% of the human gene is identical to theviral gene in the region of amino acid homology (nucleotides 563-1636 inc-erb-A, data not shown). These comparisons, coupled with previous datadescribing other human erb-A genes mapping to chromosome 17 (Jansson, etal., 1983; Spurr, et al., 1984; and Dayton, et al., 1984), indicate thatthe human placenta c-erb-A gene we have isolated is distinct. One of thetwo chromosome 17 erb-A genes (both of which we propose to call hc-erb-Aalpha) has 82% similarity with the v-erb-A gene by nucleotide sequenceand 89% by amino acid identity (See Dayton, 1984). Therefore, theplacenta c-erb-A cDNA, which we propose to call hc-erb-A beta, is moredistantly related to the viral erb-A gene than the hc-erb-A alpha genes.

Amino acid sequence comparisons between the viral and cellular erb-Aprotein products and the glucocorticoid receptor indicate graded levelsof homology with the carboxy-terminal half of the hGR (FIG. III-2). Thehighest degree of similarity is found in a cysteine-rich sequence of 65amino acids beginning at c-erb-A amino acid residue 102 (see Weinberger,et al., 1985). There is 47% amino acid identity in the comparison withthe hGR and 52% identity when c-erb-A is compared with the humanoestrogen receptor (hER) amino acid sequence (FIG. III-2). We haveproposed that this region of the hGR represents the DNA binding domain(Weinberger, et al., 1985). Mutagenesis and expression studies haveprovided direct evidence for its role in transcriptional activation (seeExperimental Section II). Regions downstream from the cysteine-richdomain, which correspond to the hormone binding domain, contain reducedyet significant (17%) homology to the hGR and hER as found previouslywith the viral erb-A product (see Weinberger, et al., 1985; Green, et al, 1986; and Greene, et al., 1986).

III. C. Multiple erb-A Genes

Hybridization of restriction endonuclease-digested human placenta DNAwith a labeled DNA fragment derived from the cysteine-rich region of thec-erb-A polypeptide (FIG. III-1a,b) produced two bands in everydigestion with the exception of PvuII (FIG. III-3a). The greaterintensity of the 9.4 kb-band suggested that it contains two hybridizingDNA fragments. When the hybridization conditions were relaxed,additional bands were observed in the products of each enzyme digestion.For example, two faint bands of 5.1 and 3.6 kb were seen after PstIdigestion (FIG. III-3b). The hybridization probe contains a singleinternal PstI site (FIG. III-1a) which probably explains the increasednumber of PstI bands detected with this probe.

Similar high-stringency hybridization experiments were performed using a260 bp EcoRI-BamHI fragment from the 5' untranslated region of pheA4(FIG. III-1a and data not shown). Two hybridizing DNA fragments weredetected with all restriction enzymes providing further support for theexistence of two related c-erb-A genes. No additional DNA bands wereseen when the hybridization was performed under relaxed conditions usingthis probe (data not shown). We conclude that there are two closelyrelated hc-erb-A beta proto-oncogenes and a third, less similar one, inthe human genome.

Laser-sorted chromosomes were prepared from human lymphoid cells (Lebo,et al., 1984), bound to nitrocellulose filters and hybridized undernon-stringent hybridization and washing conditions using the 1.5-kbEcoRI insert from pheA4 (FIG. III-3c). This probe detected only humanchromosome 3-specific DNA and suggests that the three c-erb-Abeta-related genes are chromosomally linked, although we cannot strictlyexclude the possibility that the non-stringently hybridizing erb-A genemaps in another chromosome. Interestingly, another steroidreceptor/erb-A genomic fragment has recently been identified bycharacterizing the integration site for hepatitis B virus in a humanhepatocellular carcinoma (Dejean, et al., 1986). This locus has alsobeen mapped on human chromosome 3 suggesting that the erb-A genes may beclosely linked.

III. D. Expression of c-erb-A Genes

Northern blots hybridizing cytoplasmic poly(A)-containing RNA's,isolated from various human cell lines or human term placenta, with a650-bp BamHI-PstI fragment from the pheA4 (FIG. III-1a) revealed asingle RNA species of 2,000 nucleotides that is most abundant in HeLaand MCF-7 cells (FIG. III-4a). The size of the mRNA indicates that wehave isolated a nearly full-length c-erb-A cDNA. HT1080 cells contain asmall amount of the 2-kb transcript while it is undetectable in IM-9cells. Human placenta appears to contain multiple species of 5, 3 and2.5 as well as 2.0 kb RNA. It is unclear whether the multiple placentabands represent nuclear precursors, or mature mRNA's from a single gene,or the products of other erb-A genes.

The protein products of the human c-erb-A cDNA were characterized by invitro translation. A cDNA containing the entire c-erb-A coding regionwas inserted into the EcoRI site of the expression vector pGEM3 in bothorientations. Capped RNA transcripts synthesized by T7 polymerase fromthese templates were used to program protein synthesis in rabbitreticulocyte lysates and the ³⁵ S-methionine-labeled products wereseparated on SDS-polyacrylamide gels (Laemmli, 1970). Proteins withM_(r) of 55, 52, and 35K were detected when peA101 DNA (erb-A sensetranscripts) was used as a template (FIG. III-4b, lanes 3 and 4) but notwhen peA102 DNA (erb-A antisense transcripts) was used (FIG. III-4b,lane 2). The 55 and 52K products may correspond to polypeptidesinitiating translation at methionines 1 and 27, respectively (FIG.III-1), while the 35K product may be a proteolytic breakdown product.

III. E. Thyroid Hormone Binding

Structural similarity of the steroid ligands as well as the partialamino acid sequence homology (40%) between the carboxy termini of thehGR and hER (which specify the hormone binding domains; see Krust, etal., 1986) support the hypothesis that the steroid receptors comprise afamily of regulatory proteins. The more distant homology between thecarboxy terminus of c-erb-A and the steroid receptors suggested thaterb-A proto-oncogenes probably do not encode steroid receptors, but isconsistent with the hypothesis that erb-A may respond to othermolecules. We have shown that in vitro translation can be used as ameans to characterize hormone binding activity of cloned steroidreceptors (see Experimental Section I). Accordingly, this assay wasemployed to identify the putative c-erb-A ligand.

Steroid and thyroid hormones exert their effects through fundamentallysimilar mechanisms. To address the possibility that erb-A may be thethyroid hormone receptor, the in vitro translation products were mixedwith ¹²⁵ I-3,5,3'-triiodo-L-thyronine (¹²⁵ I-T₃) in a hormone bindingreaction (see Samuels, et al., 1974). Nonspecific hormone binding wasdetermined by adding a 500-fold molar excess of cold T₃ to parallelsamples. Remarkably, the mixture containing the 55 and 52K polypeptides(peA101) acquired ¹²⁵ I-T₃ binding whereas the anti-sense RNA-programmedlysates (peA102) had only background binding. Hormone binding wassensitive to proteases but not nucleases (data not shown). Affinity ofT₃ for the cloned erb-A protein was determined by Scatchard analysis(FIG. III-5a) A K_(d) value of 5×10⁻¹¹ M was obtained, which isvirtually identical to the T₃ binding (6×10⁻¹¹ M) in HeLa cell nuclearextracts (data not shown).

Specific analogues of thyroid hormones have characteristic competitionpatterns for T₃ binding to the native thyroid hormone receptor. Wedetermined whether the erb-A product synthesized in vitro shared thesame intrinsic hierarchy of affinities for a range of natural andsynthetic thyroid hormones. Competition of ¹²⁵ I-T₃ binding was mosteffectively achieved with 3,5'; 3'-triiodothyroacetic acid (TRIAC) whichinhibited 50% of ¹²⁵ I-T₃ binding at 300 p^(M) (FIG. III-5b). Inaddition, L-thyroxine (FIG. III-5b), D-T₃ and reverse T₃(3,3',5'-triiodo-L-thyronine) competed more poorly than T₃ (FIG.III-5c), while 100 micro^(M) vitamin D₃, aldosterone, cortisol,testosterone, progesterone or oestradiol did not compete (data notshown), consistent with the biochemical properties of the rat thyroidhormone receptor (see Samuels, et al., 1974; Samuels, et al., 1979; andLatham, et al., 1976).

High salt (0.4 M KCl) HeLa cell nuclear extracts contained thyroidhormone (¹²⁵ I-T₃) binding activity, while none was found in cytoplasmicor low-salt (0.1M KCl) nuclear extracts (data not shown). Competition ofthyroid hormone binding in the nuclear extracts was quantitativelysimilar to that of lysates containing c-erb-A made in vitro (compareFIG. III-5b and d). Furthermore, thyroid hormone binding using 0.4M KClnuclear extracts from IM-9 cells, which contain undetectable levels ofc-erb-A mRNA (FIG. III-4, lane 4), is negligible when compared withsimilar HeLa extracts (data not shown). These results provide directevidence that c-erb-A is the thyroid hormone receptor.

III. F. Conclusions

The data in this section provide three criteria that identify thec-erb-A product as the thyroid hormone receptor. First, the overallstructural homology of c-erb-A is likely to be a ligand-responsiveregulatory protein. Second, the expressed protein product has the sameintrinsic hierarchy of affinities for natural and synthetic thyroidhormones as the native receptor. Third, the molecular weights of erb-Ain vitro translation products are similar to the photo-affinity-labeledrat thyroid hormone receptor (Pascual, et al., 1982). The identity oferb-A as the thyroid hormone receptor could be further substantiated bydemonstrating its transcriptional regulation of T₃ -responsive genessuch as the growth hormone gene.

Analysis of the hGR and hM has revealed the proteins to be composed of aseries of functional domains. (See Experimental Sections I and IV, andWeinberger, et al., 1985a; also see Carlstedt-Duke, et al., 1982;Dellweg, et al., 1982; Reichman, et al., 1984; and Sherman, et al.,1978.) These include a cysteine-rich region which contains structuralsimilarity to a repeated cysteine-rich region which contains structuralsimilarity to a repeated cysteine-rich sequence found in Xenopus 5S genetranscription factor IIIA and other transcriptional regulatory proteins(Miller, et al., 1985; Berg, 1985) as well as carboxy-terminalsequences, which encode the steroid-binding domain (see ExperimentalSection II and Weinberger, et al , 1985a; also see Kumar, et al., 1986).Extension of this analogy to the thyroid hormone receptor would predictits hormone binding region to be localized near the carboxy-terminal endof the molecule (FIG. III-6). Putative DNA binding sequences would befound in the cysteine-rich region (FIG. III-6) where DNA bindingproperties of the hGR and hER appear to be localized (see ExperimentalSection II, and Kumar, et al., 1986; also S. Hollenberg and R.M.E.,unpublished data).

III. G. Thyroid Hormone Receptor and Oncogenesis

Expression of the v-erb-A product in avian erythroblasts is required formaintenance of the fully transformed phenotype (see Graf, et al., 1983;Frykberg, et al , 1983; Sealy, et al., 1983; and Kahn, et al., 1986).Chickens infected with viruses lacking the v-erb-A gene display a lessvirulent disease, while in vitro these infected erythroblastsdifferentiate spontaneously and grow only with complex mediasupplements. Cells infected with erb-A⁺ /erb-B⁺ virus, however, have anincreased capacity for self-renewal and display a less differentiatedphenotype. Structural alterations of the v-erb-A protein could give riseto a product exerting aberrant properties of growth control. Forinstance, changes at the carboxyl terminus might affect thyroid hormonebinding activity, as has been shown for the beta form of the humanglucocorticoid receptor, resulting in a constitutively active molecule(see Experimental Sections II and III; also see Weinberger, et al.,1985a), where changes abolish steroid binding activity. Insertionalmutants in this domain also inactivate steroid binding properties (seeExperimental Section II). However, deletion of the hormone bindingregion gives rise to a constitutively active receptor indicating thatthis domain plays a modulatory role in transcriptional activation (V.Giguere and R.M.E,, unpublished data). These data lead us to predictthat v-erb-A is unlikely to bind hormone and is rather a constitutivelyactive form of the thyroid hormone receptor. The identification of erb-Aas the thyroid hormone receptor provides the first direct evidence of acausative involvement of enhancers and their binding proteins inoncogenic transformation.

III. H. A Superfamily of Regulatory Genes

Similarity of the steroid receptors with the v-erb-A oncogene productwas sufficient to allow us to propose that both have evolved from aprimordial receptor gene (Weinberger, et al., 1985a). Two surprisingresults have emerged from the studies presented here. The first is thatthe occurrence of a family of erb-A proto-oncogenes implies theexistence of one or more other molecules closely related to the thyroidhormone receptor. Physiological studies have not predicted the existenceof a second class of thyroid hormone receptors and thus thecharacterization of this family may shed new light on mechanisms ofdevelopmental and homeostatic regulation. The second surprisingobservation from these results is the close kinship of the thyroidhormone receptors with the steroid hormone receptor family. Thisrelationship indicates that these molecules may all be part of asuperfamily of regulatory proteins that have arisen over evolutionarytime to match the increasing developmental and physiological demands ofmore complex eukaryotes.

III. I. Detailed Description of Figures Referred to in ExperimentalSection III FIG. III-1

Restriction map and sequencing strategy (a) and nucleotide and predictedamino acid sequence (b) of human placenta c-erb-A cDNA. a, Orientationsof the two subclones pheA4 and pheA12 relative to the compositerestriction map. Common restriction endonuclease cleavage sites areabove the linear map. Thin lines, untranslated sequences; hatched box,erb-A coding region; arrows, DNA fragments sequenced. b, Nucleotidesequence of the composite erb-A cDNA is presented in the 5' to 3'orientation. The translational open reading frame related to the viralerb-a protein ²¹ is shown above the nucleotide sequence. Adenosineresidues (.sup.˜ 130) are found at the 3' end of pheA12. Numbers abovethe translated sequence indicate amino acid residues and nucleotidenumbers are on the right of the sequence.

FIG. III-1 Methods

Recombinant phage (.sup.˜ 10⁶) from each of two human placenta lambdagt10 cDNA libraries (Huynh, et al., 1985) were screened using anick-translated (Rigby, et al , 1977) 500-bp PstI fragment isolated frompAEV-11 (Vennstrom, et al.. 1980)). The hybridization mixture contained50% formamide, 1×Denhardt's, 5×SSPE, 0.1% sodium dodecyl sulphate (SDS),100 microgram ml⁻¹ denatured salmon sperm DNA and 10⁶ c.p.m. ml⁻¹ of ³²P-labeled PstI fragment (specific activity=1×10⁸ c.p.m. microgram⁻¹).Duplicate nitrocellulose filters were hybridized at 37° C. for 18 h,washed three times for 20 min each in 0.1×SSC, 0.1% SDS (1×SSC=150 mMNaCl, 15 mM trisodium citrate) and autoradiographed at -70° C. with anintensifying screen. Two hybridization-positive clones were isolated,subcloned into the EcoRI site of pUC8, and sequenced by the chemicalcleavage method (Maxam, et al., 1977).

FIG. III-2

Amino acid sequence comparison between the carboxy-terminal portions ofthe v-erb-A oncogene product, the human placenta c-erb-A polypeptide andthe human glucocorticoid and estrogen receptors. Translated amino acidsequences for both the v-erb-A protein (upper sequence) and the humanplacenta c-erb-A polypeptide (second sequence) were compared by aligningmatching residues. A computer program for the concurrent comparison ofthree or more amino acid sequences (Johnson, et al., 1986) was used toalign c-erb-A human glucocorticoid receptor (hGR third sequence fromtop; see Experimental Section I, FIG. I-2) and human oestrogen receptor(hER, bottom sequence; see Green, et al., 1986) carboxy-terminal aminoacid sequences, on the basis of progressive evaluation of selectedsegments from each sequence. Amino acid residues matched in at leastthree of the polypeptides are boxed. Amino acid matches between the twoerb-A polypeptides are indicated by an asterisk above the top sequencein each column. Amino acid identities between the steroid receptors aredesignated by crosses below the sequences. Hyphens and gaps are insertedto maximize the number of matches in the comparison. Cysteine residuesare conserved between the four polypeptides printed white-on-black.

FIG. III-3

Southern analysis and chromosome mapping of human placenta DNA withc-erb-A DNA probes. a, Human term placenta DNA was digested withrestriction endonucleases and products were separated on a 0.8% agarosegel. DNA's were transferred to nitrocellulose paper (Southern, 1975) andhybridized in 50% formamide, 5×SSPE, 1×Denhardt's, 0.1% SDS, 100microgram ml⁻¹ salmon sperm DNA with the 450-bp SstI fragment from thepheA4 which was nick-translated to a specific activity of 5×10⁸ c.p.m.microgram⁻¹. The filter was washed in 0.1×SSC, 0.1% SDS at 60° C. andexposed to X-ray film at -70° C. with an intensifying screen. LambdaHindIII DNA markers (size in kb) are aligned to the left of theautoradiogram. b, Analysis of placenta DNA using the same c-erb-A probeas in section a above, under non-stringent hybridization conditions. Aparallel blot containing the same samples was hybridized as in sectiona, except that 35% formamide was used. The hybridized filter was washedin 2×SSC, 0.1% SDS at 55° C. and exposed to X-ray film as describedabove. c, Chromosome mapping of human c-erb-A genes. Human lymphocytechromosomes were separated by laser cytofluorometry (Lebo, et al., 1984)and probed using the non-stringent hybridization conditions described insection b, but with the 1.5 kbp EcoRI insert from pheA4 as probe.

FIG. III-4

Human c-erb-A expression. a, Northern analysis of RNA's from human celllines and human placenta. Cytoplasmic poly(A)-containing RNA (12microgram) from HeLa, MCF-7 and IM-9 cells, or total poly(A) RNA's fromHT1080 or placenta, were separated on a 1% agarose gel containingformaldehyde, transferred to nitrocellulose (Thomas, 1980) and probedusing a nick-translated 650-bp BamHI-PstI pheA4 fragment. CytoplasmicRNA's were isolated from the cell lines using isotonic buffer and 0.5%NP40, while the placenta RNA's were extracted from fresh tissue usingguanidine thiocyanate (Chirgwin, et al., 1979). Lane 1, HeLa; lane 2,HT1080, lane 3, human placenta; lane 4, IM-9; lane 5, MCF-7. b,Synthesis of erb-A polypeptides in vitro. The ³⁵ S-methionine-labeledproducts synthesized using T7 polymerase-catalysed RNA transcripts wereseparated on a 12.5% SDS-polyacrylamide gel which was fluorographed (EN³HANCE, New England Nuclear). Lane 1, control (without mRNA); lane 2,peA102 (anti-sense RNA, 4 microliters); lane 3, peA101 (sense RNA, 1microliter); lane 4, peA101 (4 microliters RNA). Sizes of proteinstandards: bovine serum albumin, 66.2K; ovalbumin, 45K and carbonicanhydrase, 31K.

FIG. III-4 Methods

The EcoRI insert from pheA12 which contains the entire coding region ofc-erb-A was inserted into the EcoRI site of pGEM3 (Promega Biotec) inboth orientations. Plasmid DNA's peA101 and peA102 were linearized withHindIII, purified on 0.8% agarose gels and used as templates for T7polymerase-catalysed synthesis of RNA's in vitro (see ExperimentalSection I). After P60 chromatography, total nucleic acid material (2micrograms) was used to program protein translation in a rabbitreticulocyte lysate system (Promega Biotec) with ³⁵ S-methionine (25microCi, 1100 Ci mmol⁻¹, New England Nuclear) final volume 25microliters.

FIG. III-5

Thyroid hormone binding to erb-A polypeptides synthesized in vitro. a,Scatchard analysis of ¹²⁵ I-T₃ binding to the erb-A polypeptides made invitro. The erb-A polypeptides (2 microliters from the in vitrotranslation mixture in a total volume of 2 ml) were assayed for specificthyroid hormone binding activity using hydroxylapatite to measure theamount of bound and free labeled hormone at different concentrations of¹²⁵ I-T₃ as described (Gruol, 1980). b, Competition of thyroid hormoneanalogues for ¹²⁵ I-T₃ binding to erb-A polypeptides synthesized invitro. Samples (2 microliters) from the peA101 (sense strand)-programmedreactions were used in ¹²⁵ I-T₃ standard binding reactions (Samuels, etal., 1974) with increasing concentrations of unlabeled thyroid hormoneor analogues to compete with labeled hormone. Specifically bound thyroidhormone is plotted against concentration of competitor compound. c,Competition of triiodothyronine isomers from ¹²⁵ I-T₃ binding to erb-Apolypeptides synthesized in vitro. Binding reactions were performed asabove adding increasing concentrations of T₃ isomers. Thyroid hormonebound to erb-A is plotted on the ordinate. d, Competition of thyroidhormone analogues for ¹²⁵ I-T₃ binding to 0.4M KCl HeLa cell nuclearextracts. HeLa cell nuclei were extracted with a buffer containing 0.4MKCl (Samuels, et al., 1974). The protein extract (25 micrograms)(determined by Bio-Rad protein assay) was mixed with 0.6 nM ¹²⁵ I-T₃ instandard binding reactions with increasing concentrations of thyroidhormone and analogues (Samuels, et al., 1974; Latham, et al., 1976).

FIG. III-5 Methods

Labeled ¹²⁵ I-3,3',5-triiodo-L-thyronine (New England Nuclear, 2,200 Cimmol⁻¹, 0.3 nM final) was mixed with erb-A polypeptides synthesized inthe in vitro translation mixture (described in FIG. III-4) in T₃-binding buffer (0.25 M sucrose, 0.25M KCl, 20 mM Tris-HCl (pH 7 5), 1mM MgCl₂, 2 mM EDTA, 5 mM dithiothreitol (DTT)) (Samuels, et al., 1974)at 0° C. for 2 h in a final volume of 250 microliters. Specific hormonebinding was determined by adding a 1,000-fold excess of unlabeledhormone and assayed by counting radioactivity eluting in the excludedvolume from a Sephadex G-25 fine (Pharmacia) 0.9×4.0 cm column (Samuels,et al., 1974).

FIG. III-6

Schematic comparison of the steroid and thyroid hormone receptors.Amino-acid sequences of the receptor molecules aligned in FIG. III-2 arerepresented schematically. CYS, cysteine-rich region encoding theputaive DNA binding domain found in the receptor proteins (Cys-richregion residues are: c-erb-A, 102-169; hGR, 421-486; hER, 185-250);Cortisol oestradiol and T₃ /T₄ hormone binding regions in the carboxyltermini; 1 MM, immunogenic region of the human glucocorticoid receptor.Numbers separating boxes, percentage amino acid identities between thereceptor species in the intervals between the vertical broken lines;hGR, human glucocorticoid receptor; hER human oestrogen receptor;hc-erb-A beta, human thyroid hormone receptor.

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EXPERIMENTAL SECTION IV Cloning of Human Mineralocorticoid ReceptorComplementary DNA: Structural and Functional Kinship with theGlucocorticoid Receptor IV. A. Summary

Low-stringency hybridization with human glucocorticoid receptor (hGR)complementary DNA was used to isolate a new gene encoding a predicted107-kilodalton polypeptide. Expression studies demonstrate its abilityto bind aldosterone with high affinity and to activate genetranscription in response to aldosterone, thus establishing its identityas human mineralocorticoid receptor (hMR). This molecule also shows highaffinity for glucocorticoids and stimulates a glucocorticoid-responsivepromoter. Together the hMr and hGR provide unexpected functionaldiversity in which hormone-binding properties, target gene interactions,and patterns of tissue-specific expression may be used in acombinatorial fashion to achieve complex physiologic control.

IV. B. Introduction

The hypothalamic-pituitary-adrenal axis integrates a variety ofneuroendocrine inputs to regulate the synthesis and secretion of theadrenal corticosteroids. These steroid hormones exert effects on growth,development, and homeostasis by their interaction with intracellularreceptor proteins that directly regulate the transcription of sets oftarget genes (1,2). Two receptor systems have been defined for thecorticosteroids; these are termed the glucocorticoid receptor (GR) andthe mineralocorticoid receptor (MR). Early functional assays classifiedthe corticosteroids as either glucocorticoid, by their effect inpromoting glycogen deposition in the liver, or mineralocorticoid, bytheir effect in promoting sodium retention by the kidney. However, eachsteroid class is not restricted to interacting with only its cognatereceptor, and glucocorticoids, in particular, can have substantialmineralocorticoid activity (1-3).

It is now evident that the MR has significant in vitro affinity for bothglucocorticoids and mineralocorticoids (3,4). Since the circulatinglevels of glucocorticoids are several orders of magnitude higher thanthose of aldosterone, the primary mineralocorticoid, glucocorticoidactivation of the MR may be functionally significant. Whereas thesecretory epithelia of tissues such as kidney and intestine regulateelectrolyte and water balance in response to aldosterone, it is possiblethat additional mechanisms confer these tissues with sensitivity tomineralocorticoids (5). No clear functional role has emerged for the MRexpressed in other tissues, but physiological responses in brain mayresult from glucocorticoid interactions with the MR (5-7).

Despite the availability of high-affinity radioactively labeled ligands,the MR has been refractory to purification, and its biochemicalproperties, in comparison to GR, remain poorly understood. Applicationof the techniques of molecular biology to the study of the MR wouldfacilitate its biochemical characterization and, eventually, anunderstanding of the genes under its transcriptional control and theroles their products play in homeostasis.

Molecular cloning of the glucocorticoid (see Experimental Section I andrefs. 8,9), estrogen (10), and progesterone (11) receptors has permittedthe determination of their primary amino acid structures and predictionof functional domains common to this family of regulatory proteins.Experimental dissection of glucocorticoid (see Experimental Section IIand ref. 12) and estrogen (13) receptors has revealed a centrallylocated DNA-binding domain rich in cysteine, lysine, and arginine, and acarboxyl-terminal region where steroid hormones interact. Functionalstudies of GR suggest that hormone binding to the carboxyl terminusunmasks the DNA binding region to permit interactions of receptor withDNA and activation of transcription (14, 15). Comparison of thecysteine-rich DNA-binding regions of steroid and thyroid hormonereceptors shows a high degree of relatedness between these molecules(16). The invariant cysteine residues have led to the hypothesis thatcoordination of Zn²⁺ metal atoms maintains a structural configurationfor DNA binding analogous to that proposed for Xenopus 5S genetranscription factor IIIA (17). The steroid-binding regions of thesteroid receptor family also show substantial conservation consistentwith evolution of various receptor classes from a common ancestralprecursor (11, 16).

We have used the structural similarity between steroid hormone receptorsto isolate a gene product closely related to the human glucocorticoidreceptor (hGR). Nonstringent hybridization with an hGR probe was used toisolate a human genomic DNA fragment highly related to the hGRcysteine-rich sequence. Using this DNA as a probe, we obtainedcomplementary DNA's (cDNA's) that code for a molecule having a stronghomology with the hGR from the cysteine-rich region to the carboxylterminus When expressed in cells, this molecule binds aldosterone withhigh affinity and activates aldosterone-response transcription of thelong terminal repeat (LTR) of the mouse mammary tumor virus (MMTV). Theoverlap of the ligand and DNA sequence specificities of this humanmineralocorticoid receptor with those of hGR suggest that the distinctroles traditionally assigned to these regulatory molecules should bereconsidered.

IV C. Isolation of hMR cDNA

For the identification of glucocorticoid receptor-related genes, humanplacenta DNA was digested with restriction endonucleases, fractionatedby agarose gel electrophoresis, and the fractions were hybridized withhGR 1.2, an 1100-bp fragment of hGR cDNA containing sequences encodingthe DNA-binding domain (see Experimental Section I; also ref. 15).Southern blot analysis revealed several distinct bands specific tolow-stringency hybridization conditions (compare FIG. IV-1, A and B).The 2.5-kilobase pair (kbp) HindIII fragment (bracketed by asterisks inFIG. IV-1B) was well resolved from other hybridizing bands and wasjudged suitable for direct genomic cloning. HindIII-digested DNA fromhuman placenta was preparatively size-fractionated on an agarose gel,and the 2.5-kbp region was isolated for the construction of a genomiclibrary. This lambda gt10 library was then screened under conditions oflow-stringency hybridization with hGR 1.2 as the probe. The insert fromone positive genomic clone, lambda HGH, was nick-translated and used asa probe on a Southern blot under high-stringency hybridizationconditions (FIG. IV-1C). The 2.5-kbp HindIII signal corresponded to thatseen under nonstringent conditions, indicating that a portion of thedesired genomic fragment had been isolated. Sequence analysis of theinsert from lambda hGH revealed an exon of 140 base pair (bp) flanked byintron sequences (FIG. IV-1D). Overall this exon has 68 percentnucleotide identity with the homologous hGR cDNA sequence, but a regionconserving 85 nucleotides out of 104 probably confers itscross-hybridization properties. This highly conserved region correspondsto a portion of the hGR DNA-binding domain (15). The lambda HGH exoncodes for 46 amino acids beginning with 16 nonconverted residues andfollowed by the first of the highly conserved cysteine residuescharacteristic of steroid hormone receptors (8-11). Of the next 30residues, 28 are identical to hGR. These analyses demonstrated theisolation of a genomic fragment containing a sequence related to, butclearly distinct from, that found in the hGR cDNA sequence (seeExperimental Section I).

The insert from lambda HGH was used as a probe to screen cDNA librariesfor clones corresponding to this hGR-related gene. Mineralocorticoidreceptor was considered a candidate to be encoded by such a gene. Sincekidney is known to be a mineralocorticoid-responsive tissue, severalhuman kidney cDNA libraries were screened. Eleven positive clones wereisolated from these lambda gt10 libraries at a frequency of three tofour per 10⁶ recombinant phage. Two overlapping clones, lambda hk2 andlambda hk10, were subjected to nucleotide sequence analysis and togetherfound to span 5823 nucleotides (FIG. IV-2). The exon-intron boundariesof lambda HGH were verified by sequencing these cDNA clones. The lambdahk10, encompassing nucleotides 1 to 3750, contains a large open readingframe predicting the entire primary amino acid sequence. The DNA insertfrom lambda hk2 extends from nucleotides 802 to 5823, but contains aninternal 351-bp deletion from 2235 to 2586. Three additional clones wereexamined and determined to have the same structure as lambda hk10 in thedeleted region. It is likely that the deletion in lambda hk2 representseither a cloning artifact or a rare messenger RNA (mRNA) splicing error(18). The sequence of the reported 3'-untranslated region downstream ofnucleotide 3750 is derived from lambda hk2. The composite sequence ofthese two cDNA's is termed hMR (FIG. IV-2A). With the first in-frame ATG(position 223) downstream of an in-frame termination codon (position136), hMR has a 5'-untranslated region of at least 216 nucleotides. Thesequence surrounding this first ATG agrees with the consensus describedby Kozak (19). This predicted initiator methionine codon begins an openreading frame encoding 984 amino acids. Following a termination codon(position 3175) is a 2.6-kb 3'-untranslated region with a typicalpolyadenylation signal (AATAAA) found 17 nucleotides upstream of a70-nucleotide poly(A) (polyadenylated) tract. Long 3'-untranslatedregions are a characteristic feature of steroid hormone receptor mRNA's(see Experimental Section I; also refs. 9-11).

IV. D. The DNA- and Hormone-Binding Regions

The protein encoded by hMR cDNA has the structural properties of asteroid hormone receptor closely related to hGR. Comparison of thepredicted amino acid sequence of hMR with that of hGR demonstrated highdegrees of homology with both the hGR DNA binding and steroid bindingdomains. The hMR gene encodes a protein of 984 amino acids with apredicted molecular size of 107 kD, significantly larger than the 777residues of hGR. This size discrepancy is primarily due to the largeamino terminus, which bears no homology to hGR. Considerableheterogeneity of size and sequence for this region exists between thereceptors for glucocorticoid, estrogen, and progesterone (seeExperimental Section I; also refs. 9-11). Amino acid homology begins inthe centrally located DNA region with 94 percent amino acid identity in68 residues (FIG. IV-3). Separating the DNA-binding domain and thecarboxyl-terminal steroid-binding domain is a region with relatively lowsequence conservation found between other steroid hormone receptors. Ithas been speculated that the region may serve as a molecular hingebetween the two domains (see Experimental Section II and ref. 13).Comparison with hGR shows this region of hMR to contain an additional 24amino acids including a sequence of 4 glutamines followed by 8 prolinesencoded by repetitive nucleotide elements. The significance of thisunusual sequence in terms of origin and function is unclear, butstructure-breaking prolines are consistent with a hinge region. Acomparison of the carboxyl-terminal 250 amino acids of hMR with hGRshows 57 percent amino acid identity as well as a number of conservativeamino acid substitutions. Some of these substitutions may preservehydrophobic regions necessary for steroid hormone interaction.

IV. E. Expression and Hormone

We have used transfection of the monkey kidney cell line CV1 and itsderivative (that is, SV40 T antigen-transformed) cell line COS-1(referred to as COS) to study glucorticoid receptor function. (SeeExperimental Section II.) High levels of polypeptide expression fromtransfected hMR were essential to facilitate steroid-binding experimentsin transfected cells. Since plasmids containing the SV40 origin ofreplication can replicate to high copy numbers in COS cells, anexpression vector for hMR coding sequences similar to pRShGR alpha, usedpreviously in hGR studies, was constructed. The plasmid, pRShMR,contains the hMR coding sequence, under the control of the promoter fromRous sarcoma virus, and the SV40 origin of replication (FIG. IV-4A).

Ligand specificity of the hMR protein was determined by preparingcytosol extracts from COS cells transfected with pRShMR Two days aftertransfection, cells were harvested, and hormone binding was measured bya dextran-treated charcoal assay. Mock-transfected control extracts hadno specific binding activity for [³ H]aldosterone, whereas extracts frompRShMR-transfected cells found significant amounts of [³ H]aldosteronewith high affinity. A dissociation constant (K_(D)) of 1.3 nM for thebinding of [³ H]aldosterone was determined by a Scatchard analysis (FIG.IV-4B). This value is in good agreement with those reported foraldosterone binding to mineralocorticoid receptor (2, 20). Competitionexperiments were then performed to examine the ability of differentunlabeled steroids to compete with 5 nM [³ ]aldosterone for binding whenpresent at 1-, 10-, or 100-fold molar excess (FIG. IV-4, C and D). Thisprovided a measure of the relative affinity of each of these steroidsfor hMR. The results of these experiments show that aldosterone,corticosterone, deoxycorticosterone, hydrocortisone (cortisol) all havevery similar affinities for hMR. Dexamethasone, progesterone, andspironolactone demonstrated weaker binding affinity while estradiolcompeted very poorly for binding to hMR. Overall, this hierarchy ofaffinities indicated that hMR encoded the human mineralocorticoidreceptor (2, 20).

Steroid hormone action is characterized by hormone-dependent modulationof target gene transcription. The assay for transcriptional regulationby transfected hGR in CVI cells (see Experimental Section II) wasadapted to hMR (FIG. IV-5). The expression plasmid used forsteroid-binding assays, pRShMR, was cotransfected with a reporterplasmid called GMCAT, which contains the MMTV LTR linked to thebacterial gene for chloramphenicol acetyltransferase (CAT). Thus CATactivity provides an enzymatic assay for the transcriptional activity ofthe MMTV promoter. The MMTV promoter contains several glucorticoidresponse elements (GRE's), enhancer-like DNA sequences that conferglucocorticoid responsiveness via interaction with the GR (21). It waspossible that hMR, because of the near identity of its DNA-bindingdomain to that of hGR, might also recognize the MMTV LTR. When CV1 cellswere cotransfected with pRShMR and GMCAT, we observed full CAT activity.This activity was independent of added aldosterone suggesting that, incontrast to transfected hGR, sufficient hormone was present in serum(fetal calf serum, 5 percent) to fully activate hMR (FIG. IV-5B). In thepresence of charcoal-treated serum (22) CAT activity became responsiveto the addition of exogenous aldosterone (FIG. IV-5C), indicating thathMR cDNA encodes a functional steroid hormone receptor. While the hMRwas also activated by the glucocorticoid agonist dexamethasone, the hGRdid not respond to even supraphysiological concentrations (10 nM) ofaldosterone.

IV. G. Tissue-Specific Expression

We examined the expression of MR mRNA homologous to hMR cDNA in rattissues by Northern blot hybridization (23). Classical mineralocorticoidtarget tissues such as kidney (24) and gut (25), as well as tissues suchas brain, pituitary, and heart, contained mRNA homologous to hMR (FIG.IV-6). Aldosterone-sensitive cells in kidney are primarily restricted tothe distal and cortical collecting tubules (2), and therefore a modestlevel of expression in this tissue was not unexpected. High levels of MR(type I corticosteroid-binding sites) have been reported in rat brain,particularly in the hippocampal formation (4, 6). In comparing dissectedhippocampal RNA with RNA prepared from total brain, we found a strikingenrichment of message in the hippocampus. While aldosterone binding hasbeen reported for pituitary (26), cultured aortic cells (27), and spleen(28), no such activity has been reported in muscle. Liver expresses GR,but has no detectable high-affinity aldosterone-binding activity (29),and as would be expected no hybridization to liver RNA was observed.Reprobing of the same Northern blot with an analogous portion of hGRcDNA demonstrated hybridization to mRNA species of different sizes, andindicated that the MR and GR do show differential patterns oftissue-specific expression.

IV. H. Chromosome Mapping

To determine the chromosomal location of the mineralocorticoid receptorgene, we tested hMR against a panel of rodent-human somatic cell hybridsretaining different combinations of human chromosomes (30). The DNAfragments specific for the mineralocorticoid receptor gene segregatedconcordantly with human chromosome 4 in 15 hybrid cell lines. Discordantsegregation was observed for all other human chromosomes, includingchromosome 5, site of the glucocorticoid receptor gene (see ExperimentalSection I and ref. 31). To confirm the assignment to chromosome 4, wetested a restricted set of microcell hybrids, each of which carry one tothree human chromosomes (32), for the hMR gene by Southern analysis(FIG. IV-7). Six EcoRI fragments detected by the coding portion oflambda hk2 co-segregate with chromosome 4 in this hybrid panel. Inparticular, the hMR gene is present in HDM-1132B, a cell line thatcarries chromosome 4 as its only human chromosome.

IV. I. Implications for Adrenal Corticosteroid Physiology

Human mineralocorticoid receptor cDNA encodes a polypeptide that ishighly homologous to the human glucocorticoid receptor. In theDNA-binding domain, hMR maintains approximately 94 percent amino acididentity to hGR while the steroid-binding domain localized in thecarboxyl terminus has 57 percent identity. The recently reportedsequence (11) of the rabbit progesterone receptor (rPR) also has a highdegree of relatedness to hMR. Comparison of the amino acid identity inhGR and rPR structural domains with that of hMR (FIG. IV-8) demonstratesthe remarkable similarity of these functionally distinct regulatoryproteins. The homology of hMR with rPR is almost identical to thehGR-hMR comparison, with 90 percent of the amino acids shared in theDNA-binding domain and 56 percent in the steroid-binding region. Incontrast, a comparison of the same regions of hMR with human estrogenreceptor (10) indicates 56 percent identity in the DNA binding domainand 21 percent sequence identity in the steroid-binding carboxylterminus. The degree of structural homology shared by hMR, hGR, and rPR,and the structural relatedness of their ligands, suggests that they maycomprise a subfamily of steroid hormone receptors.

Expression of the hMR polypeptide in COS cells by transient transfectionpermitted the evaluation of its steroid-binding potential. The resultsof these analyses indicated that hMR cDNA encodes a humanmineralocorticoid receptor. Scatchard analysis demonstrated thatextracts from cells transfected with pRShMR bound [³ H]aldosterone witha K_(D) of 1.3 nM, while reported K_(D) values for aldosterone bindingto MR range from 0.5 to 3 nM (2). This is the single most importantcriterion in defining this gene product as the human mineralocorticoidreceptor. Steroid-binding competition studies have further supportedthis identification of hMR. The mineralocorticoid deoxycorticosteroneand the glucocorticoids corticosterone and cortisol compete aseffectively as aldosterone itself, whereas the synthetic glucocorticoiddexamethasone and progesterone have lower affinities for the hMR.

The extensive amino acid sequence identity in the presumptivesteroid-binding domains of hMR, hGR, and rPR is compatible with thesimilar ligand-binding properties of these receptors. Themineralocorticoid, glucocorticoid, and progesterone receptors exhibit alimited ability to discriminate between the similar 21-carbon atomstructures of the mineralocorticoids, glucocorticoids, and progestins.This lack of specificity is particularly relevant to the MR and GR. Forexample, the MR binds glucocorticoids with an affinity equal to that foraldosterone. Indeed, it may be that only in tissues such as kidney,where additional mechanisms confer selective response to aldosterone,does the MR function as a classical mineralocorticoid receptor (3, 5).The MR also binds progesterone with a high affinity, but one lower thanits affinity for corticosteroids. There is some indication thatprogesterone may act as a partial agonist or antagonist ofmineralocorticoid action (33), and it is not clear whetherglucocorticoids act as full agonists in binding to the mineralocorticoidreceptor. Similarly, the GR binds glucocorticoids with a K_(D) between20 to 40 nM and it binds aldosterone with a K_(D) between 25 to 65 nM(2). Therefore, the important distinction between the hormone-bindingproperties of MR and GR may not be one of ligand specificity, but ratherof a high-affinity versus a lower affinity receptor for thecorticosteroids.

The function of the hMR in vivo is complicated by the serumcortisol-binding protein, transcortin. This protein sequesters cortisoland, because of its differential distribution, transcortin couldinfluence local glucocorticoid concentration. High levels of transcortinin kidney would reduce available cortisol from plasma to favoraldosterone sensitivity, whereas low levels of transcortin in the brainwould suggest that, in the central nervous system, glucocorticoids maybe the predominant hMR ligand. Thus, the preferred physiologic ligandfor hMR apparently varies depending on the site of receptor expression(3). This model and others (5) have been proposed to explain theresponsiveness of some tissues to aldosterone despite much higher levelsof competing glucocorticoids.

The degree of homology between hMR and hGR in the DNA-binding domain(only four amino acid residues differ in this conserved 68-residueregion) suggests that these receptors may recognize similar regulatoryelements. The activation of the MMTV LTR by the transfected hMR inresponse to both aldosterone and dexamethasone supports this conclusion,although the progesterone receptor has also been demonstrated toregulate this promoter (21). Furthermore, differences between hMR andhGR in the DNA-binding domain, or in other regions such as the highlydivergent amino termini of these molecules, may influence target genespecificity in ways not revealed in this assay. However, we haveutilized transcriptional regulation of the MMTV LTR by hMR and hGR toexamine their activation by mineralocorticoids and glucocorticoids.While the hMR response was approximately equivalent with either 10 nMaldosterone or dexamethasone, hGR was activated by dexamethasone but wasinsensitive to aldosterone in this assay. Transcriptional activation byhMR in response to exogenous cortisol was also observed. These dataindicate that in transfected cells both mineralocorticoids andglucocorticoids can activate hMR-mediated gene transcription. On thebasis of this functional property, we conclude that the hMR is highlyresponsive to adrenal corticosteroids and therefore may function as aglucocorticoid receptor.

In addition to elucidating the pharmacologic and physiologic function ofthe mineralocorticoid receptor in coordinating response tocorticosteroids, the isolation of hMR cDNA will facilitate investigationof the role of hMR in a number of disease states, among themhypertension and pseudohypoaldosteronism (PHA). An association ofmineralocorticoids with hypertension has been recognized for severaldecades, and it may be that hMR-mediated sodium retention and increasedblood volume are, in part, responsible for some forms of hypertension(34). PHA is an autosomal recessive disorder characterized by lack ofresponsiveness to normal or elevated aldosterone levels. Recent work hasdemonstrated diminished or complete loss of high-affinityaldosterone-binding sites in patients with this disease (35) which islikely to result from a mineralocorticoid receptor genetic defect. Thechromosomal mapping of the hMR gene suggests the PHA locus should resideon chromosome 4.

Cloning and expression of functional hMR has provided unexpected insightand should stimulate new interest into the mechanisms underlyingphysiologic complexity, and allow the development and testing of newmodels for the coordinate regulation of gene networks.

IV. J. Detailed Description of Figures Referred to in ExperimentalSection IV FIG. IV-1

Isolation of a genomic sequence related to the hGR gene. (A)High-stringency Southern analysis of human placenta DNA digested withthe indicated restriction endonucleases. hGR cDNA (hGR1.2) was used as aprobe. Sizes of lambda DNA fragment markers (in kilobase pairs) preparedby HindIII digestion are indicated next to the autoradiogram. (B)Low-stringency Southern analysis. The 2.5-kbp band bracketed byasterisks in the HindIII lane was the sequence targeted for directgenomic cloning. (C) Isolation of this genomic sequence in a clonedesignated lambda HGH is demonstrated by its use as a probe on a similarSouthern blot. The lambda HGH genomic fragment contains the hybridizinginternal EcoRI fragment isolated from this cloning. (D) The intron-exonstructure of the lambda HGH genomic fragment and its homology with hGR.The hGR-related exon found within lambda HGH is boxed in black with itspredicated amino acid sequence. Conserved cysteine residues areindicated with white dots. Portions of intron sequence with consensussplice donor and acceptor sites underlined are shown flanking the exon.Nucleotide homology with the hGR is shown underneath. Nucleotide numbersfor hGR are from FIG. I-2, discussed in Experimental Section I; also seeHollenberg, et al. (1985) (ref. 8) for publication of the study usedherein as Experimental Section I. For Southern analysis, we digested DNAfrom human term placenta with restriction endonucleases, and productswere separated on a 0.8 percent agarose gel. The DNA's were transferredto nitrocellulose paper and hybridized under either stringent ornonstringent conditions. Stringent hybridization was performed with 50percent formamide, 5×SSPE (NaCl, NaH₂ PO₄, EDTA, pH 7.4), 1×Denhardt's,0.1 percent SDS, salmon sperm DNA at 100 microgram/ml, and probe (10⁶cpm/ml) at 42° C. For nonstringent hybridization, 35 percent rather than50 percent formamide was used. Washing conditions consisted of 0.1×SSC(standard saline citrate) with 0.1 percent SDS at 60° C. for stringentanalyses and 2×SSC with 0.1 percent SDS at 55° C. for nonstringentfilters. Washing conditions with the 338-bp inset from lambda HGH asprobe were modified to 2×SSC with 0.1 percent SDS at 68° C. Forisolation of lambda HGH, human placenta DNA (300 microgram) was digestedwith HindIII and size-fractionated on a 1 percent low-melting agarosegel (Seaplaque, FMC). The gel was sliced in 0.5-cm strips, and the DNAwas purified by phenol extraction and ethanol precipitation. DNA (2microgram) from the fraction corresponding in size to the band bracketedby asterisks in (B) was repaired with Klenow DNA polymerase for EcoRIlinker addition. After digestion with EcoRI and removal of excesslinkers on a Sepharose 4B column, this DNA was ligated to EcoRI-digestedlambda gt10 DNA and packaged in vitro (lambda arms and extracts fromVector Cloning Systems, San Diego, Calif.). About 4×10⁵ independentrecombinants were screened under conditions identical to those used forthe nonstringent Southern analysis to obtain lambda hGH.

FIG. IV-2

Nucleotide sequence and primary amino acid structure of humanmineralocorticoid receptor. (A) Composite structure of hMR aligned witha line diagram of some restriction endonuclease cleavage sites (EcoRIsites shown at nucleotides I and 5823 and derived from linkers). Thecomposite was assembled from two overlapping lambda gt10 clones, lambdahk10 and lambda hk2. Parentheses in the line diagram of lambda hk2indicate a 351-bp deletion. The hatched box indicates predicted codingsequence with initiator and termination condons indicated. (B) Completenucleotide sequence of hMR and its predicted primary amino acidsequence. Underlined are a 5' in-frame termination codon upstream of thepredicted initiator methionine and four potential polyadenylation sites(AATAAA). Human kidney lambda gt10 libraries (18) were screened with theinsert from lambda HGH under the same conditions described for Southernanalysis under high-stringency conditions with this probe. Overlappingdeletions of each cDNA were obtained (36) by the Cyclone rapid deletionsubcloning method (International Biotechnologies). Deletion clones weresequenced by the dideoxy procedure (37), and any gaps or ambiguitieswere resolved by the chemical cleavage method (38). DNA sequences werecompiled and analyzed by the programs of Devereux, et al. (39) andStaden (40).

FIG. IV-3

Amino acid homology of mineralocorticoid receptor with glucocorticoidreceptor. The primary amino acid sequence of hMR has been aligned withthat of hGR for maximum homology by introducing gaps as indicated bydots. Numbers were taken from FIG. IV-2 for hMR and from FIG. I-2 forhGR. No significant homology was found upstream of the region shown.Vertical lines indicate identical amino acid residues. Arrows showputative boundaries of the DNA-binding (DNA) and steroid-binding(Steroid) domains. The amino-terminal border of the DNA-binding domainwas arbitrarily defined by the first conserved cysteine residue whilethe carboxyl-terminal limit was chosen on the basis of mutagenesisstudies which indicated sequences necessary for DNA-binding andtranscriptional activation (15). Several conserved basic residues thatfollow the DNA binding domain may also be important for these functions.The limits of the steroid-binding domain, while defined by the region ofamino acid homology, are also consistent with mutational analysis.Single letter abbreviations for the amino acid residues are: A, Ala; C,Cys; D, Asp; E, Glu; F, Phe, G, Gly; H, His; I, Ile; K, Lys; L, Leu; M,Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; andY, Tyr.

FIG. IV-4

Steroid-binding properties of expressed hMR. (A) Structure of pRShMR,the hMR expression plasmid (41). (B) Scatchard analysis of tritiatedaldosteone binding in extracts prepared from pRShMR-transfected COScells. Each point was assayed in triplicate with 100 micrograms ofextract protein in a 200-microliter incubation at 0° C. for 2.5 hours.The nonspecific binding determined with a 500-fold excess of unlabeledaldosterone was approximately 20 percent of total counts. No specificbinding was seen in mock-transfected cells. (C and D) Competition ofunlabeled steroids for binding with 5 nM [³ H]aldosterone in transfectedCOS cell extracts. The results of two independent trials representativeof these competition experiments are shown. Cold competitor was presentin 1-, 10-, or 100-fold molar excess. The value for 100 percent bindingwas determined by subtracting the number of counts per minute bound inthe presence of 1000-fold excess of unlabeled aldosterone from thecounts bound in the absence of competitor. Abbreviations: Aldo,aldosterone, Doc, deoxycorticosterone; Dex, desamethasone; Spiro,sprionolactone; E21, 17 beta-estradiol; CS, corticosterone; HC,hydrocortisone; and Prog, progesterone. Subconfluent COS cells weretransfected by the DEAE-dextran method (42) with 10 micrograms of pRShMRper dish. Cells were maintained for 2 days in DMEM (Dulbecco'smodification of Eagle's minimum essential medium) with 5 percentcharcoal-treated fetal calf serum, then harvested [in 40 mM tris-HCl (pH7.8), 10 mM NaCl, 1 mM EDTA, 10 mM Na₂ MoO₄, 5 mM dithiothreitol,antipain (5 microgram/ml), leupeptin (5 microgram/ml), and 500 microMphenylmethylsulfonyl fluoride. After centrifugation at 15,000×g for 10minutes, extracts were adjusted to 100 mM NaCl and 5 percent glycerolbefore binding. Labeling reactions with [³ H]aldosterone (specificactivity 78 Ci/mmol, Amersham) were incubated for 2.5 hours at 0° C. ina total volume of 200 microliters, and then for 10 minutes with 20microliters of 50 percent dextran-coated charcoal (10:1 activatedcharcoal:dextran). After centrifugation at 15,000×g for 2 minutes at 4°C., tritium in supernatant was quantified by liquid scintillationspectrophotometry.

FIG. IV-5

Transcriptional activation of MMTV LTR by hMR and hGR expressionplasmids in transfected CVI cells. (A) Structure of GMCAT. This plasmidwas cotransfected with the steroid receptors as a reporter gene forhormone-dependent transcriptional activation (see Experimental SectionII). (B) Differential CAT enzyme activity found after hMr or hGRtransfection with normal serum. Transfected cells were maintained inDMEM with 5 percent fetal calf serum. Serum was treated with charcoal toeliminate free steroids in subsequent experiments so that the effects ofexogenous steroids could be determined. (C) Differential induction ofCAT activity by aldosterone or dexamethasone in cells transfected withhMR or hGR. CVI cells were cotransfected with 10 micrograms of eitherpRSVgal (control), pRShMR, or pRShGR alpha and 10 micrograms of thereporter GMCAT and cultured in the absence (-) or presence of 10 nMaldosterone (A) or 10 nM dexamethasone (D). AC, 3-acetylchloramphenicol;C, chloramphenicol. Two days after transfection by calcium phosphatecoprecipitation (43), extracts were prepared for CAT assay (44). Theassays were incubated for 6 hours with 50 micrograms of protein extract.

FIG. IV-6

Northern analysis of mineralocorticoid receptor mRNA's in rat tissues.The 1270-bp EcoRI fragment (1770 to 3040) from lambda hk10 was used as aprobe for the expression of homologous mRNA's in rat. Ten micrograms ofpoly(A)⁺ mRNA were used in all lanes. Migration of ribosomal RNA's (28Sand 18S) are indicated for size markers. After hybridization understringent conditions, the filter was washed twice for 30 minutes eachtime in 2×SSC with 0.1 percent SDS at 68° C.

FIG. IV-7

Chromosomal localization of hMR gene by Southern analysis of microcellhybrids. The construction and characterization of these hybrids has beenpreviously described (32). The human chromosome content of each is asfollows: HDm-4A (chromosome 20), HDm-5 (chromosome 14 and an unspecifiedE group chromosome), HDm-9 (chromosomes 20, 14, and 21), HDm-15(chromosomes 21, 11, and 4), HDm-20 (chromosomes 7 and 14), andHDm-1132B (chromosome 4 only). Human (HeLa) and mouse (3T#) control DNAsamples are also shown. Genomic DNA from microcell lines (10 micrograms)was digested with EcoRI and subjected to electrophoresis through a 1.0percent agarose gel, transferred to a nylon membrane (Nytran, Schleicher& Schuell), and hybridized with a hMR cDNA probe under high-stringencyconditions (FIG. IV-1). The radioactive probe was synthesized by theKlenow fragment of Escherichia coli DNA polymerase from two randomlyprimed (45) hMR cDNA templates (the 1000- and 800-bp EcoRI fragments oflambda hk2). The sizes of HindIII-digested lambda DNA fragments areindicated next to the autoradiogram.

FIG. IV-8

Schematic amino acid comparisons of the hGR, hMR, and rPR structures.Primary amino acid sequences have been aligned schematically with thepercentage amino acid identity indicated for each region of homology inthe intervals between dotted lines. The amino acid position of eachdomain boundary is shown for each receptor. N and C represent the aminoand carboxyl termini, respectively. Cys corresponds to the cysteine-richregion encoding the putative DNA-binding domain while Steroid (cortisol,aldosterone, or progesterone) designates the steroid-binding domain. Theimmunogenic region (IMM) of the hGR is also indicated. Amino acidresidue numbers are taken from (see Experimental Section I) for hGR,Loosfelt, et al. (11) for rPR, and from our data for hMR.

IV. K. References Referred to in Experimental Section IV

1. Baxter, J. D., and Tyrrell, J. B., in Endocrinology and Metabolism,Felig, P., Baxter, J. D., Broadus, A. E., and Frohman, L. S., Eds., pp.385-510, McGraw-Hill, New York (1981); Marver, D., in BiochemicalActions of Hormones, Litwack, G., Ed., Vol. 12, pp. 385-431, AcademicPress, Orlando Fla. (1985).

2. Fanestil, D. D., and Park, C. S., Annu. Rev. Physiol., 43:637 (1981).

3. Funder, J. W., in Adrenal Cortex, Anderson, D. C., and Winter, J. S.D., Eds., pp. 86-95, Butterworths, London (1985).

4. Beaumont, K., and Fanestil, D. D., Endocrinology, 113:2043 (1983;Krozowski, Z. S., and Funder, J. W., Proc. Natl. Acad. Sci., USA,80:6056 (1983).

5. Funder, J. W., and Sheppard, K., Annu. Rev. Physiol, 49:397 (1987).

6. Reul, J. M. H. M., and deKloet, E. R., Endocrinology, 117:2505(1985).

7. McEwen, B. S., deKloet, E. R., and Rosterne, W., Physiol. Rev.,66:1121 (1986).

8. Hollenberg, S. M., et al., Nature, 318:635 (1985).

9. Miesfeld, R., et al., Cell, 46:389 (1986).

10. Green, S., et al., Nature, 320:134 (1986); Greene, G. L., et al.,Science, 231:1150 (1986).

11. Loosfelt, H., et al., Proc. Natl. Acad. Sci., USA, 83:9045 (1986).

12. Giguere, V., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M.,Cell, 46:645 (1986).

13. Kumar, V., Green, S., Staub, A., and Chambon, P., EMBO J., 5:2231(1986); Green, S., and Chambon, P., Nature, 235:75 (1987).

14. Godowski, P. J., Rusconi, S., Miesfeld, R., and Yamanoto, K. R.,Nature 325:365 (1987).

15. Hollenberg, S. M., Giguere, V., Segui, P., and Evans, R. M., Cell,49:39 (1987).

16. Weinberger, C., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R.M., Nature, 318:670 (1985); Krust, A., et al., EMBO J., 5:891 (1986);Sap, J., et al., Nature, 324:635 (1986); Weinberger, C., Hollenberg, S.M., Rosenfeld, M. G., and Evans, R. M., Nature, 318:641 (1985).

17. Miller, J., McLachlan, A. D., and Klug, A., EMBO J., 4:1609 (1985).

18. Deletions in other cDNA clones have been reported in these libraries[G. I. Bell, et al., Nucleic Acids Res., 14:8427 (1986)].

19. Kozak, M., Nature, 308:241 (1984).

20. Armanini, D., Strasser, T., and Weber, P. C., Am. J. Physiol.,248:E388 (1985).

21. Ringold, G. M., Yamamoto, K. R., Bishop, J. M., and Varmus, H. E.,Proc. Natl. Acad. Sci., USA, 74:2879 (1977); Payvar, F., et al., Cell,35:381 (1983); Schiedereit, C., Geisse, S., Westphal, H. M., and Beato,M., Nature, 304:749 (1983).

22. For the removal of endogenous steroids, washed charcoal was added toserum [2 g (dry weight) per 100 ml] and incubated at 37° C. for 40minutes then at 55° C. for 30 minutes. Charcoal was removed byfiltration through 0.45 then 0.2 microm filters.

23. No human cell lines have been reported to express high-affinityaldosterone-binding sites. Northern blot analysis of human ovary RNArevealed two hybridizing RNA species approximately sized at 6 and 4 kb.No aldosterone-binding activity has been reported in ovary.

24. Fuller, P. J., and Funder, J. W., Kidney Int., 10:154 (1976);Matulich, D. T., Spindler, B. J., Schambelan, M., and Baxter, J. D., J.Clin. Endocrinol. Metab., 43:1170 (1976).

25. Pressley, L., and Funder, J. W., Endocrinology, 97:588 (1975).

26. Lan, N. C., Matulich, D. T., Morris, J. A., and Baxter, J. D.,Endocrinology, 109:1963 (1981).

26. Krozowski, Z., and Funder, J. W., Endocrinology, 109:1221 (1981).

27. Meyer, W. J., and Nichols, N. R., J. Steroid Biochem., 14:1157(1981); no type I receptors were detectable in an early study in heart[Funder, J. W., Duval, D., and Meyer, P., Endocrinology, 93:1300 (1973).

28. Swaneck, G. E., Highland, E., and Edelman, J. S., Nephron, 6:297(1969).

29. Duval, D., and Funder, J. W., Endocrinology, 94:575 (1974).

30. Dracopoli, N. C., et al., Am. J. Hum. Genet., 37:199 (1985); Rettig,W., et al., J. Exp. Med., 162:1603 (1985).

31. Gehring, U., Segnitz, B., Foellmer, B., and Francke, U., Proc. Natl.Acad. Sci., USA, 82:3751 (1985).

32. Lugo, T. G., Handelin, B. L., Killary, A. M., Housman, D. E., andFournier, K., Mol. Cell. Biol., in press.

33. Wambach, G., and Higgins, J. R., Endocrinology, 102:1686 (1978).

34. Vallotton, M. B., and Favre, L., in Adrenal Cortex, Anderson, D. C.and Winter, J. S. D., Eds., pp. 169-187, Butterworths, London (1985).

35. Armanini, D., et al., N. Engl. J. Med., 313:1178 (1985).

36. Dale, R. M. K., McClure, B. A., and Houchins, J. P., Plasmid, 13:31(1985).

37. Sanger, F., Nicklen, S., and Coulson, A. R., Proc. Natl. Acad. Sci.,USA, 74:5463 (1977).

38. Maxam, A. M., and Gilbert, W., Methods Enzymol., 65:499 (1980).

39. Devereux, J., Haeberli, P., and Smithies, O., Nucleic Acids Res.,12:387 (1984).

40. Staden R., Nucleic Acids Res., 10:4731 (1982); Staden, R., NucleicAcids Res., 12:521 (1984).

41. Construction of pRShMR. The 3.75-kb insert from lambda hk10 wasligated into the EcoRI site of pGEN4 (Promega) oriented with the mp18polylinker adjacent to the 5' end of hMR coding sequence. Digestion ofthis plasmid (phk10) with HindIII generated at HindIII fragment spanningthe polylinker site to the hMR site at position 3562; this fragment wasisolated and the ends were repaired with the Klenow fragment of DNApolymerase I. Plasmid pRSVCAT [C. M. Gorman, G. T. Merlino, M. C.Sillingham, I. Pastan, B. H. Howard, Proc. Natl. Acad. Sci., USA,79:6777 (1982)] was digested with HindIII and HpaI, and the HindIII-HpaIfragment containing pBR322 sequences, the RSV LTR, and the SV40polyadenylation site was also repaired. Ligation of the hMR fragment tothe fragment from pRSVCAT yielded a vector which, in the correctorientation, has hMR coding sequence driven by the RSV promoter. Sitesbracketed in FIG. IV-4A were lost in this cloning step. To improvetranslational efficiency several upstream initiation and terminationcodons in the 5'-untranslated region were deleted by digesting thevector with AccI to remove an .sup.˜ 200-bp sequence from the mp18polylinker to position 188 in the hMR 5'-UT region. Finally, anNdeI-linkered SV40 origin of replication was introduced into the NdeIsite (12) to generate pRShMR.

42. Deans, R. J., Denis, K. A., Taylor, A., and Wall, R., Proc. Natl.Acad. Sci., USA, 81:1292 (1984).

43. Wigler, M., et al., Cell, 16:777 (1979).

44. Gorman, C. M., Moffat, L. F., and Howard, B. H., Mol. Cell. Biol.,2:1044 (1982).

45. Feinberg, A. P., and Vogelstein, B., Anal. Biochem., 137:266 (1984).

EXPERIMENTAL SECTION V Identification of a New Class of Steroid HormoneReceptors V. A. Introduction

The gonads and adrenal glands produce a large variety of steroidsclassified into five major groups which include the estrogens,progestins, androgens, glucocorticoids and mineralocorticoids. Gonadalsteroids control the differentiation and growth of the reproductivesystem, induce and maintain sexual characteristics and modulatereproductive behavior. Similarly, adrenal steroids influencedifferentiation in addition to their vital roles as metabolicregulators. Despite this wide range of physiological actions, theeffects of each steroid rest primarily upon the specific cognatereceptors which it binds, and therefore steroid hormone action might bemore precisely classified according to the receptors that mediate theirbiologic action. The successful cloning, sequencing and expression ofthe human glucocorticoid receptor (hGR) cDNA (see Experimental SectionI, which was published as ref. 1), soon followed by those encoding theestrogen²,3 (hER), progesterone⁴ (hPR), and mineralocorticoid (hMRreceptors) (see Experimental Section IV, which was published as ref. 5),plus homologues from various species⁶⁻¹¹, provide the firstopportunities to study receptor structure and the molecular mechanismsby which these molecules modulate gene expression. Sequence comparisonand mutational analysis of these proteins reveal structural featurescommon to all classes of steroid hormone receptors (see ExperimentalSection II which was published as ref. 12; also see refs. 13 and 14). Inparticular, the receptors share a highly conserved cysteine rich region,now referred to as the DNA-tau domain, that contains all the informationrequired for both DNA-binding and trans-activation functions of theglucocorticoid receptor¹⁵,16. The presence of a common segment betweenreceptors provides the possibility of scanning the genome for relatedgene products. For example, hMR cDNA was isolated by using the hGR as ahybridization probe (see Experimental Section IV, which has beenpublished as ref. 5). One way that molecular biology can advance ourunderstanding of health and human disease and the physiology thatgoverns these events is through the identification of new hormoneresponse systems. In this study, using the highly conserved DNA-tauregion of the human estrogen receptor cDNA as a hybridization probe, wehave isolated two cDNA clones encoding polypeptides that comprise thestructural features of the steroid hormone receptors.

V. B. cDNA Clones for Receptor hERR1

One approach to search for unrecognized hormone response systems is tosystematically employ reduced stringency hybridization to screenrecombinant DNA libraries for novel hormone receptors. The DNA-tausegment of the estrogen receptor was used to initiate these studies.Analysis of a lambda gt10 human testis cDNA library identified 3positives at a frequency of one clone per 3×10⁵ recombinant phages.Nucleotide sequence analysis revealed that two of these clones actuallyencode the estrogen receptor while the third one, spanning 2.0 kilobasesand named lambda hT16, showed only partial sequence homology. In turn,this clone was used to screen human fetal kidney and adult heart cDNAlibraries, resulting in the identification of 3 additional clones. Bothclones from the kidney library, lambda hKE4 and lambda KA1, representthe same gene product as lambda hT16 while the cardiac clone, lambdahH3, is only partially related. The composite sequence of the threecDNA's sharing identical sequences, herein referred to as hERR1, isshown in FIG. V-1. Assuming a poly(A) tail of .sup.˜ 150-200nucleotides¹⁷, this sequence (.sup.˜ 2430 nt) must be nearly fulllength. The cDNA insert from lambda hKA1 contains nucleotide 179 to 2430while lambda hKE4 represent a rare messenger RNA splicing error withdeletion of exon 2 and insertion of intron sequences. The exon/intronboundaries suggested by lambda hKE4 were confirmed by cloning andpartially sequencing the genomic fragments encoding this gene (data notshown). The sequence surrounding the first ATG agrees with the concensusdescribed by Kozak¹⁸ for a translation initiation site. An open readingframe of 521 amino acids predicting a polypeptide of Mr 57300 is flankedby a 775 nucleotide 3'-untranslated region.

V C. cDNA Clone for Receptor hERR2

The characterization of clone lambda hH3 reveals it to encode a uniquepolypeptide highly related to hERR1. FIG. V-2 shows the 2153 nucleotidesequence of lambda hH3 and the primary structure of the protein productdesignated hERR2. The translation initiation site was assigned to themethionine codon corresponding to nucleotides 100-102 because this isthe first ATG triplet that appears downstream from an in-frameterminator TGA (nucleotides 31-33). An open reading frame containing 433amino acids encodes a polypeptide of Mr 47600 and is followed by a3'-untranslated region of 752 nucleotides.

V. D. Characterization of hERR1 and hERR2

As mentioned earlier, steroid hormone receptors are composed of distinctfunctional domains

that can be identified by sequence analysis¹⁴. The predicted hERR1 andhERR2 polypeptides contain the expected domain features of steroidreceptors. Amino acid comparison between hERR1 and hERR2 shows thatthese two proteins have divergent amino termini and that no homology canbe detected with other classes of receptor within this region (data nowshown). This finding is in agreement with previous comparison studies(see Experimental Section IV; also see refs. 5,8,10,14) which showedthis region to be hypervariable in sequence. Alignment of thecarboxy-terminal region of hERR1, hERR2, hER and hGR (FIG. V-3) showsthat the highest degree of homology between these proteins is found in acysteine-rich region of 66 amino acids, corresponding to the DNA-taudomain (see Experimental Section II; also see ref. 15) of the steroidhormone receptors, located between amino acid 175 and 240 of hERR1.There is a 91% amino acid identity in the comparison of hERR1 withhERR2, 68% with hER and 56% with hGR. The positions of the 9 cysteineresidues are strictly conserved but the absence of a histidine residueat position 206 of hERR1 and position 134 of hERR2 marks a majordifference with the previously described steroid hormone receptors. Itwas originally thought that this histidine residue might be involvedwith the conserved cysteine residues in the formation of a DNA-bindingfinger. The recent demonstration that this histidine residue is alsoabsent in the corresponding amino acid sequence of the vitamin Dreceptor¹⁹, another member of the ligand-binding transactivation factorsuperfamily, suggests that Zn²⁺ atoms interact exclusively with cysteineresidues in order to coordinate the formation of the proposedDNA-binding fingers present in those proteins. The putative steroidbinding domain, positioned between amino acid 295 and 521 of hERR1,shows 63% identity when compared to hERR2, 36% to hER and 28% to hGR.

V. E. Tissue Distribution of mRNS's for hERR1 and hERR2

Steroid receptors are expressed in characteristic tissue specificpatterns that directly correlate to their primary physiologic effects.Perhaps, the distribution of these putative receptors would provide aclue to their hidden identity. Accordingly, total RNA isolated from avariety of rat and human tissues was fractionated onformaldehyde-agarose gel electrophoresis and transferred tonitrocellulose filters. Using lambda hKA1 as a probe, a 2.6 Kb mRNAencoding hERR1 was detected in all rat and human tissues analyzed, withsurprisingly high levels in the cerebellum and hippocampus and thelowest levels seen in the liver, lungs, seminal vesicles and spleen(FIG. V-1A). Thus, it appears that the hERR1 gene is widely andabundantly expressed, although present in much higher levels in the ratcentral nervous system. In contrast to the hERR1 mRNA expressionpattern, the distribution of the mRNA encoding the hERR2 protein isrestricted to a few specific tissues where very low levels of mRNA canbe detected (FIG. V-4B). Using a probe derived from the clone lambdahH3, a 4.8 Kb mRNA was detected in kidney, heart, testis, hypothalamus,hippocampus, cerebellum and rat prostate. However, no hybridizationcould be detected in the human placenta or prostate. Considering thedifference in exposure time and the resulting signal intensity, levelsof hERR2 mRNA are approximately 10 to 100-fold lower than that of hERR1.

V. F. Homology between hERR1 AND hERR2

Prior studies indicate that the degree of homology of the ligand bindingdomains between the steroid hormone receptors reflects the structuralrelatedness of their ligands. For example the hGR, hMR and hPR, whichshow 56% identity in their ligand binding domains (see ExperimentalSection IV), bind closely related hormones. Indeed the hMR bindsglucocorticoids with an affinity equal to that of aldosterone, and alsobinds progesterone with relatively high affinity (see ExperimentalSection IV). In the case of the hERR gene products, amino acid sequencehomology reveals a relatively more distinct relationship with hER, 70%in the DNA-tau region and 36% in the steroid binding region (FIG. V-5).These levels of homology are lower than those observed between hGR, hMRand hPR and thus predict that the putative hERR proteins interact with aclass of steroid hormones distinct from the estrogens. However, thehomology between hERR1 and hERR2 suggests that they are receptors foreither a single or two closely related steroid metabolites. Preliminarysteroid binding studies using the products of in vitro translation ofcapped SP6 RNA produced from hERR1 and hERR2 coding sequences orexpression of the two cDNA's in COS-1 cells (see Experimental SectionsIV and II) have failed to demonstrate binding of any major classes ofsteroids, including estrogens and androgens.

V. G. Conclusion

The tissue distribution of hERR1 and hERR2 mRNA's expression suggeststhat each putative receptor will control distinct biological functions.How might the functions of these steroid hormone receptors have beenoverlooked? Most likely many of their activities have erroneously beenattributed to other receptors with differences being classified asatypical effect. The recent identification of neuronal steroids²⁰,21provides evidence for new steroid hormones with possible paracrineactions within the brain. Such systems could have easily escapedprevious physiological detection. Thus, the isolation of two novelsteroid hormone receptor cDNA's marks the first step toward identifyinga new hormone response system.

V. H. Detailed Description of Figures Referred to in ExperimentalSection V FIG. V-1

Restriction map (A) and DNA sequence and predicted amino-acid sequence(B) of hERR1. A, The composite cDNA for hERR1 is represented at the top,with noncoding (thin line) and coding (stippled portion) sequencesindicated. Common 6-nucleotide restriction enzyme sites are drawn abovethe linear map. Overlapping cDNA inserts used to determine the sequenceare shown The wavy line near the 5' end of lambda hKE4 indicatesdivergent sequence. B, Nucleotide sequence of the composite hERR1 cDNAwith the deduced amino acids given above the long open reading frame.

FIG. V-1 Methods

The clone lambda hT16 was isolated from a human testis lambda gt10 cDNAlibrary (Clonetech) using a nick-translated²² 446-bp BglI-BamHI fragmentisolated from pER945, a linker-scanning mutant¹² derived from the hERcDNA (V. Giguere and R. M. Evans, unpublished data). The hybridizationmixture contained 35% formamide, 1×Denhardt's, 5×SSPE, 0.1% sodiumdodecyl sulfate (SDS), 100 micrograms ml⁻¹ denaturated salmon sperm DNAand 10⁶ c.p.m. ml⁻¹ of ³² P-labeled BglI-BamHI fragment (>10⁸cpm/microgram). Duplicate nitrocellulose filters were hybridized at 42°C. for 16 h, washed three times for 20 min each in 2×SSC, 0.1% SDS(1×SSC=150 mM Nacl, 15 mM sodium citrate) at 55° C. and autoradiographedat -70° C. with an intensifying screen. The clones lambda hKE4 andlambda hKA1 were isolated from a human kidney lambda gt10 cDNA library²³using the nick-translated insert from lambda hT16. For this screening,the hybridization mixture was modified to 50% formamide and washingconditions to 2×SSC with 0.1% SDS at 68° C. The cDNA clones weredigested with a number of restriction enzymes and the resultingfragments were subcloned in both orientations into the M13 sequencingvectors mp18 and mp19 and sequenced by the dideoxy procedure²⁴, and anygap or ambiguities were resolved by the chemical cleavage method²⁵. DNAsequences were compiled and analyzed by the programs of Devereux, etal.²⁶ and Staden²⁷.

FIG. V-2

Restriction map (A) and DNA sequence and predicted amino-acid sequence(B) of hERR2. A, Schematic representation of hERR2 cDNA; some commonrestriction enzyme sites are indicated. The stippled box represents thepredicted open reading frame. B, The complete nucleotide sequence oflambda hH3 is shown with the predicted amino acid sequence given abovethe long open reading frame. A short open reading frame in the 5'untranslated region is shown in bold type.

FIG. V-2 Methods

The clone lambda hH3 was isolated from a human heart lambda gt11 cDNAlibrary (gift from Dr. L. A. Leinwand, Albert Einstein Col. of Med.)using a nick-translated 700-bp EcoRI-SmaI fragment representing the 5'portion of lambda hKA1. Hybridization and washing conditions andsequencing strategies were as described in FIG. V-1 for the screening ofthe human kidney library.

FIG. V-3

Amino acid sequence comparison between the carboxy-terminal regions ofhERR1, hERR2, the human estrogen and glucocorticoid receptors. The fouramino acid sequences were aligned for maximum homology by introducinggaps as indicated by hyphens. Numbers were taken from FIGS. V-1 and V-2for hERR1 and hERR2, from FIG. I-2 for hGR and Green, et al.² for hER.Amino acid residues matched in at least three of the polypeptides areboxed. The asterisk above residue 206 of hERR1 indicates the position ofthe histidine residue present in the hER sequence but absent in bothhERR1 and hERR2 sequences.

FIG. V-4

Northern blot hybridization analysis of hERR1 (A) and hERR2 (B) mRNA'sin rat and human tissues.

FIG. V-4 Methods

Total RNA was isolated from various tissues using guanidinetiocyanate²⁸, separated on 1% agarose-formaldehyde gel, transferred tonitrocellulose, and hybridized under stringent conditions using anick-translated EcoRI-SmaI fragment from lambda hKA1 (a) and anick-translated 1192-bp EcoRI-HindIII fragment from lambda hH3 (b).Twenty micrograms of total RNA was used in all lanes. Migration ofribosomal RNA's (28S and 18S) are indicated for size markers. Thenitrocellulose filters were autoradiographed at -70° C. with anintensifying screen for 24 h (a) and 6 days (b). Apparent difference inmigration rate of the mRNA in (a) is an artifact from the gel.

FIG. V-5

Schematic amino acid comparisons between hERR1 and hERR2, hER, hGR andhuman thyroid hormone receptor (hT₃ R beta). Amino acid sequences havebeen aligned schematically according to the functional domain structureof the steroid and thyroid hormone receptors superfamily¹⁴. Thepercentage of amino acid identity of each receptor with hERR1 isindicated inside each domain. The amino acid position of each domainboundary is shown for each receptor.

V. I. References Referred to in Experimental Section V

1. Hollenberg, S. M., et al., Nature, 318:635-641 (1985).

2. Green, S., et al., Nature, 320:134-139 (1986).

3. Greene, G. L., et al., Science, 231:1150-1154 (1986).

4. Misrachim, M., et al., Biochem. Biophys. Res. Comm., 143:740-748(1987).

5. Arriza, J. L., et al., Science, 237:268-275 (1987).

6. Miesfeld, R., et al., Cell, 46:389-399 (1986).

7. Danielsen, M., Northrop, J. P., and Ringold, G. M., EMBO J.,5:2513-2522 (1986).

8. Krust, A., et al., EMBO J., 5:891-897 (1986).

9. Maxwell, B. L., et al., Mol. Endocrinol., 1:25-35 (1987).

10. Loosfelt, H., et al., Proc. Natl. Acad. Sci. USA, 83:9045-9049(1986).

11. Weiler, I. J., Lew, D., and Shapiro, D. J., J. Mol. Endocrinol.,1:355-362 (1987).

12. Giguere, V., Hollenberg, S. M., Rosenfeld, G. M , and Evans, R. M.,Cell, 46:645-652 (1986).

13. Kumar, V., Green, S., Staub, A., and Chambon, P., EMBO J.,5:2231-2236 (1986).

14. Evans, R. M., Science, (in press).

15. Hollenberg, S. M., Giguere, V., Segui, P., and Evans, R. M., Cell,49:39-46 (1987).

16. Miesfeld, R., Godowski, P. J., Maler, B. A., and Yamamoto, K. R.,Science, 236:423-427 (1987).

17. Sawiki, S. G., Jelinek, W., and Darnell, J. E., J. Mol. Biol.,113:219-235 (1977).

18. Kozak, M., Nature, 308:241-246 (1984).

19. McDonnell, D. P., et al., Science, 235:1214-1217 (1987).

20. Le Goascogne, C., et al., Science, 237:1212-1215 (1987).

21. Hu, Z. Y., Bourreau, E., Robel, P., and Baulieu, E. E., Proc. Natl.Acad. Sci. USA, (in press).

22. Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. J., J. Mol.Biol., 113:237-251 (1977).

23. Bell, G. I., et al., Nucleic Acid Res., 14:8427-8446 (1986).

24. Sanger, F., Nicklen, S., and Coulson, A. R., Proc. Acad. Sci. USA.74:5463-5467 (1977).

25. Maxam, A., and Gilbert, W., Proc. Natl. Acad. Sci. USA, 74:560-564(1977).

26. Devereux, J., Haeberli, P., and Smithies, O, Nucleic Acid Res.,12:387-395 (1984).

27. Staden R., Nucleic Acid Res., 10:2951-2961 (1982).

28. Chirgwin, J. M., Przybyla, A. F., McDonald, R. J., and Rutter, W.F., Biochemistry, 18:5294-5299 (1980).

EXPERIMENTAL SECTION VI A c-erb-A Binding Site in the Rat Growth HormoneGene Mediates Transaction by Thyroud Hormone VI. A. Introduction

The substance 1,3,3-triiodothyronine (T₃) stimulates growth hormone genetranscription in rat pituitary tumor cells¹⁻⁴. This stimulation isthought to be mediated by the binding of nuclear T₃ receptors toregulatory elements 5' to the transcriptional start site⁵⁻⁸.Understanding of the mechanism by which thyroid hormone activates genetranscription has been limited by failure to purify nuclear T₃ receptorsbecause of their low abundance, and by the absence of defined T₃receptor-DNA binding sites affecting T₃ regulation. Recently, human andavian c-erb-A gene products have been shown to bind thyroid hormone withhigh affinity (see Experimental Section III, which has been published asref. 10; also see ref 9) and to have a molecular weight and nuclearassociation characteristic of the thyroid hormone receptor. In thepresent study, we describe the development of an avidin-biotin complexDNA-binding assay which can detect specific, high-affinity binding ofrat pituitary cell T₃ receptors to the sequence 5'CAGGGACGTGACCGCA3',located 164 base pairs 5' to the transcriptional start site of the ratgrowth hormone gene. An oligonucleotide containing this sequencetransferred T₃ regulation to the herpes simplex virus thymidine kinasepromoter in transfected rat pituitary GC2 cells, and specifically boundan in vitro translation product of the human placental c-erb-A gene. Thedata provide supporting evidence that human c-erb-A gene mediates thetranscriptional effects of T₃ and also that GC2 cell nuclear extractscontain additional factors that modify the binding of pituitary T₃receptors to the rat growth hormone gene T₃ response element.

VI. B. Characterization of Rat Growth Hormone DNA Sequences Necessaryfor T₃ Regulation (a) Rat GH gene deletion assays

To identify the cis-active element in the growth hormone (GH) 5'flanking genomic sequence necessary for T₃ regulation we used a seriesof 5'-deleted fragments of the rat GH gene, fused to the bacterialchloramphenicol acetyltransferase (CAT) gene and transfected into ratpituitary GC2 cells (FIG. VI-1a). A 5' deletion to -235 to thetranscriptional start (CAP) site transferred regulation to T₃ (4.6-fold,FIG. VI-1a), equivalent to constructions containing 1.7 kilobases (kb)or 307 base-pairs (bp) of 5'-flanking rat GH information (data notshown), in agreement with previous studies⁵,6. (Nucleotide positions arenumbered relative to the CAP site, negative position numbers being 5' toit.) Deletions containing less than 235 bp of 5'-flanking GH informationcould not be assayed for T₃ induction because the levels of CATexpression in the absence of T₃ were not significantly above background.To overcome this problem, a rat prolactin enhancer gene containing 181and 110 bp of 5' flanking information. The 5'-deleted fragment extendingto position -181 gave 2.6-fold induction by T₃ and further deletion toposition -107 from the CAP site abolished T₃ regulation (FIG. VI-1a).

(b) CAT enzymatic assay

To assay 3' deletions, fragments of the rat Gh gene were fused to theherpes simplex virus thymidine kinase (HSV tk) promoter. A fragment ofthe Gh gene extending from position -235 to position -45 from the CAPsite produced 2.5- and 2.3-fold stimulations of CAT activity when fusedto the tk promoter in native and inverse orientation, respectively (FIG.VI-1b). A 90-bp fragment extending from positions 235 to 145 bp from theGH CAP site transferred T₃ regulation even more efficiently than the235-245 bp fragment, and suggests that a negative T₃ regulatory elementmight be located between 45 and 145 bp from the CAP site (FIG. 1a). CATmessenger RNA was analyzed by a primer extension technique ¹² todetermine whether or not the T₃ -dependent stimulation of CAT activitywhich was observed in cells that had been transfected with plasmidscontaining the tk promoter resulted from an increase in appropriatelyinitiated transcripts. The 235-245 bp GH fragment fused to the tkpromoter gave an increase of about 4-fold of correctly initiated CATmRNA in the presence of T₃ (FIG. VI-1c), a result consistent with theobserved CAT activity measurements.

(c) Avidin-biotin complex DNA-binding assay (ABCD assay)

To define further the sequences needed for T₃ regulation, it wasnecessary to document specific binding of nuclear T₃ receptors to thegrowth hormone T₃ regulatory element. Because attempts to map the T₃receptor binding site by gel shift and footprinting assays wereinitially unsuccessful, a new assay to detect specific binding wasdevised: the avidin-biotin complex DNA-binding (ABCD) assay.Double-stranded oligonucleotides were prepared containing the 5'flanking region of the GH gene necessary for T₃ regulation, withbiotin-11-dUTP at various positions, as shown in FIG. VI-2a. Initially a77-bp oligonucleotide (G209-146), containing the genomic growth hormonesequence from positions -209 to -146, was used. T₃ receptors from GC2nuclear extracts were labeled to high specific activity with ¹²⁵ I-T₃and incubated with this biotinylated oligonucleotide. Protein-DNAcomplexes were precipitated from solution after the binding reaction,using streptavidin conjugated to agarose beads. It can be seen in FIG.VI-2b that probe G209-146 resulted in the precipitation of 6,900 c.p.m.of ¹²⁵ I-T₃ activity. This represents the binding of about 3.2 fmol ofT₃ receptor and accounts for .sup.˜ 40% of the total T₃ receptor presentin the binding reaction Precipitation of ¹²⁵ I-T₃ -labeled receptors wasprobe-dependent; <15% of total precipitated ¹²⁵ I-T₃ radioactivity wasrecovered in the absence of G209-146. Addition of a 100-fold molarexcess of unlabeled T₃ reduced the precipitated ¹²⁵ I-T₃ to backgroundlevels (FIG. VI-2b), indicating that the T₃ binding protein that wasbeing precipitated by the probe was present in limited amounts. Theequilibrium binding constant for the T₃ receptor-DNA complex wasestimated to be 1.4×10⁻⁹ M (data now shown).

To investigate whether the precipitation of labeled T₃ receptors byG209-146 was dependent on specific rat GH sequences, a series ofbiotinylated probes were prepared that had no apparent sequencesimilarity to the growth hormone enhancer but which were the same lengthas G209-146 and contained the same number of biotin-11-dUTP residues. Asshown in FIG. VI-2b, the addition of 100 fmol of each probe to GC2nuclear extracts gave no measurable precipitation of ¹²⁵ I-T₃. Thisindicates that the precipitation of ¹²⁵ I-T₃ by G209-146 is dependent onthe rat GH sequence contained in the probe.

(d) Footprinting analysis

Although early attempts to localize a T₃ receptor binding site usingconventional footprinting techniques were unsuccessful, variation ofbuffering conditions in the ABCD binding assay suggested that afootprint might be achieved with crude nuclear extracts if salt and pHconditions for DNA binding were optimal (data not shown). End-labeledfragments of the GH enhancer were incubated with GC2 nuclear extractsand digested with DNase I. PAGE analysis of the digest under denaturingconditions (FIG. VI-3) gave two footprints, described previously¹³,14 ;one of these is shown, together with a 16-bp protected region in theantisense strand between nucleotides -179 and -164. This sequence, whichin the sense strand corresponds to 5' CAGGGACGTGACCGCA 3', is containedin the oligonucleotide probe shown to specifically bind T₃ receptors,and corresponds in position to a previously identified T₃ -dependentDNase-I-hypersensitive site¹⁹. A clear footprint could not be detectedin the sense strand itself, mainly because of incomplete digestion byDNase I in this G-rich region.

VI. C. Functionality of T₃ Oligonucleotide

To examine the function of this sequence in T₃ regulation of the GHgene, site-directed mutagenesis was used to delete 11 bp of thefootprint from the wild-type enhancer (mutant G delta 166/177). Additionof T₃ to cells transfected with mutant G delta 166/177 fused to the tkpromoter had no effect on the amount of CAT expression, although thewild-type enhancer stimulated CAT expression ninefold (FIG. VI-1b).Thus, removal of the putative T₃ receptor binding region, identified byoligonucleotide and DNAse I binding assays, abolished the ability of theGH enhancer to confer T₃ regulation to the tk promoter.

To demonstrate that the 16 base pairs from positions -164 to -179constituted a functional T₃ regulatory element, a double-strandedoligonucleotide was prepared containing this sequence with seven and sixbases flanking the 5' and 3' ends respectively. This oligonucleotide wasinserted in its native orientation proximal to the tk promoter(construction G29TK, FIG. VI-1b). CAT expression was stimulated 2.9-foldby T₃ in cells transfected with this construction. Insertion of threetandem repeats of this sequence (construction G293TK) resulted in afive-fold stimulation by T₃.

The short oligonucleotide used for transfer of T₃ regulation to the tkpromoter (G186-158) also specifically bound nuclear T₃ receptor bindingsite in G209-146, but this was discounted using an oligonucleotidecontaining the rat GH sequence from nucleotide positions -177 to -235that failed to bind measurable amounts of T₃ receptors (data not shown).Non-biotinylated G209-146 and G186-158 were also used to compete for thebinding of T₃ receptors with biotinylated G209-146; the relativeaffinity of T₃ receptors for G209-146 was two- to three-fold higher thanG186-158 (data not shown). The apparent decrease in affinity for theshorter probe could result from a lack of bases to participate directlyin the T₃ receptor binding reaction. This is unlikely, because thelimits of the DNase I footprint lie within the ends of this probe.Alternatively, GC2 nuclear extracts could contain other factors thatstabilize the binding of T₃ receptors to the longer probe.

VI. D. Summary and Conclusions

These experiments demonstrated that 29 base pairs of the GH gene,containing a 16-bp footprint extending from position -164 to position-179 5' to the CAP site, bound the T₃ receptor, were necessary for T₃regulation of the rat GH enhancer, and could transfer T₃ regulation tothe tk promoter. To test whether human c-erb-A also binds to thiselement, an in vivo translation product was labeled with ³⁵ S-methionine(see Experimental Section III). The product migrated as a doublet ofrelative molecular mass (M_(r)) 48,000 (48K) and 52K on SDS gelelectrophoresis and bound T₃ with a k_(d) of 5×10⁺¹¹ (data not shown).The binding of the human c-erb-A in vitro translation product to theG209-146 and G186-158 oligonucleotide probes containing the rat GH T₃regulatory element is shown in FIG. VI-4. Both the long and short probesbound the in vitro translation product significantly, but probes lackinghomology to the T₃ receptor binding site of The GH gene, such as P-EGF,did not. Based on the estimated specific activity of [³⁵ S]methioninepresent in the in vitro translation mixture, the binding activity shownin FIG. VI-4 corresponds to 1-2 fmol of erb-A protein. Unlike GC2-cellnuclear extracts containing ¹²⁵ I-T₃ receptors, the c-erb-A in vitrotranslation product was bound to the same extent by the long and shortoligonucleotides. Similar data were obtained using an in vitrotranslation product of human c-erb-A labeled with ¹²⁵¹ -T₃ (data notshown). These results indicate that the c-erb-A gene productspecifically binds to the identical T₃ regulatory sequence that is boundby T₃ receptors from GC2 nuclear extracts. They provide further evidencethat the function of the c-erb-A gene product is to mediate thetranscriptional effects of T₃.

Flug, et al. (1987) recently reported that the GH sequence betweennucleotide positions -210 and -181 was essential for the fullstimulatory effect of T₃ in transiently transfected GC cells, and alsopointed out that this region possessed limited similarity to other T₃-regulated genes. Our experiments locate the T₃ receptor-DNA bindingsite between 164 and 177 bp from the CAP site, and also confirm thefunctional importance of the sequence between positions -210 and -181 inT₃ regulation of the GH gene. This could reflect an increased affinityof the T₃ receptor for fragments of the GH enhancer which contain thisupstream element in vivo, as we observed in the in vitro DNA bindingstudies using crude nuclear extracts as a source of T₃ receptor. Thatthe erb A in vitro translation product binds comparably to G209-146 andG186-158 is consistent with the possibility that crude GC2 nuclearextracts contain an additional factor(s) that binds to the sequencebetween positions -210 and -181 and stabilizes the binding of the T₃receptor to its cognate binding site. Cooperative interactions betweeneukaryotic transcription factors is well-established¹⁶⁻¹⁸ ; in somecases this reflects the ability of one factor to alter the DNA-bindingaffinity of another. Such interactions could be important in thetissue-specific regulation of thyroid hormone action⁵,6. The ABCDbinding assay used here should be useful in addressing these questions,and it is also potentially applicable to any DNA-binding protein thatcan be selectively radiolabeled, either with a labeled ligand, bychemical modification or by in vitro translation with labeled aminoacids.

VI. E. Detailed Description of Figures Referred to in ExperimentalSection VI FIG. VI-1

Thyroid hormone responsiveness of various gene fusions containing rat GH5'-flanking sequences. a, Responsiveness of 5' and 3' deletions of therat GH gene. 5'-deleted fragments of the rat GH gene were fused to theCAT gene in a pSV2CAT-based vector¹⁸, in which the SV40 origin ofreplication and promoter were removed. These constructions weretransfected into GC2 cells and assayed for responsiveness to T₃. Becauseof low levels of basal expression of the nucleotide position -107 to +8and -181 to +8 fragments, the rat prolactin enhancer (Prl)¹¹, which isnot regulated by T₃, was placed proximal to these elements. Theillustrated 3'-deleted fragments were fused to a tk promoter fragment,extending from position -197 to position +54 and placed proximal to theCAT gene in the same vector. b, Functional analysis of the putative T₃receptor binding site. Mutant G delta 177/166 contains a deletion of 11bases of the T₃ receptor binding site from 177-166 base pairs from theCAP site. Plasmids G29TK and G293TK contain the 28-bp region of the ratGH gene that binds the T₃ receptor in one and three copies,respectively. The effect of T₃ was determined by dividing the percentageconversion of chloramphenicol in the presence of 10⁻⁹ M T₃ by thepercentage conversion in the absence of T₃. Triplicate plates weremaintained in 10% fetal calf serum stripped of T₃ by ion exchangechromatography for two days before transfection with test plasmids usingDEAE-dextran¹¹. The cells were treated with hormone 24 h aftertransfection and assayed for CAT activity after 24 h of T₃ exposure.Error limits represent the standard error of the mean. Each constructionwas assayed two to five times. A_(n) represents SV40 polyadenylationsites. c, Messenger RNA transcription initiation site analysis. Thediagram indicates the 33-nucleotide primer complimentary to nucleotides67 to 89 of the CAT coding sequence used to determine the CAP site oftranscripts of plasmids containing the tk promoter. GC cells weretransfected with test plasmids and hormone treated as described for theexperiments presented in panels a and b. Primer extension analysis wasperformed on 50 micrograms total RNA. Lanes A and B represent theextension product from cells transfected with a plasmid containing theGH fragment extending from positions 235 to 45 from the CAP site fusedto the tk promoter. Lanes C and D represent the extension product fromcells transfected with a plasmid containing the tk promoter alone. Noextension products were observed. The products shown in lanes A and Cwere from cells incubated in the absence of T₃ and those in lanes B andD were from cells treated with T₃ at a concentration of 10⁻⁹ M. Lane Eshows a HindIII digest of pBR322, used for size calibration (innucleotides).

FIG. VI-1 Methods

Construction of CAT expression vectors containing 5'-flanking sequencesof the rat GH gene from -1.7 kb to +8 bp of the CAP site has beendescribed¹¹. Fusions containing the rat prolactin enhancer were made byexcising this fragment (corresponding to rat prolactin sequencenucleotide positions 1831-1530) from plasmid pPSS¹¹ and inserting it inreverse orientation proximal to the 5' deletions of the growth hormoneelement. Fusions containing the HSV tk promoter were made by excisingfragments of the GH enhancer from the plasmid GPO (ref. 11) andinserting them into the BamHI and SalI sites of pSV2CAT-based expressionvectors proximal to the tk promoter at positions -197 to +54.Alternatively, the GH enhancer was placed proximal to the promoter atpositions -107 to +54 in a pUC8-based vector²⁰ by insertion into theBamHI and SalI polylinker sites. Site-directed mutagenesis of the GHenhancer element was performed by inserting the fragment from 235-245 bpfrom the CAP site into the BamHI and SalI sites of M13 mp18. A 21-baseoligonucleotide was synthesized which corresponded to antisense GHnucleotides -188 to -157, in which nucleotides -177 to -166 were deletedand replaced by an A nucleotide. This oligonucleotide was used to deletebases -177 to -166 in the GH enhancer using the procedure of Kunkel²¹.CAT activity was determined by radioassay of methylated chloramphenicolderivatives after thin layer chromatography¹⁹. Primer extension was bythe method of Elsholtz, et al.¹²

FIG. VI-2

Binding of T₃ receptors to oligonucleotide probes containingbiotin-11-dUTP. a, Schematic representation of two oligonucleotideprobes used to assay T₃ receptor binding to GH 5'-flanking sequences.Heavy lines, synthesized oligonucleotides with complementary 3' ends.Fine lines, bases incorporated by filling the 5' overhangs using thelarge fragment of Escherichia coli DNA polymerase. Asterisks,biotin-11-dUTP residues Restriction sites for BamHI and BglII are alsoshown. G209-146 and G186-158 contain rat GH enhancer sequences with theillustrated 5' and 3' boundaries. b, Precipitation of ¹²⁵ I-T₃ labeledT₃ receptors from GC2 nuclear extracts by various oligonucleotide probescontaining biotin-11-dUTP P-EGF, PB1-B, PB2-B, and PB4-B areoligonucleotides of 68, 53, 55 and 58 base pairs containing 10, 11, 10and 10 biotin-11-dUTP's respectively. These oligonucleotides contain5'-flanking sequences of the rat prolactin gene that lack apparenthomology to the rat GH sequence contained in G209-186. Precipitationswere performed using 100 femtomole of each probe. Background,representing ¹²⁵ I activity associated with streptavidin agarose beadsin the absence of a biotinylated olignoucleotide probe, was 1,400 c.p.min this experiment. Results are plotted as the mean and standard errorof triplicate points. The experiment is representative of sixexperiments examining the sequence specificity of ²⁵ I-T₃ binding.

FIG. VI-2 Methods

Isolated nuclei were prepared from rat GC2 cells according to thetechnique of Dingham, et al.²² and salt extracted in 0.6M KCl, 10 mMHepes, pH 7.9, 0.5 mM dithiothreitol (DTT), 0.2 mM EGTA, 20 microM ZnCl₂for 30 min on ice. The nuclear extract was desalted by gel filtration inbuffer A (50 mM KCl, 20 mM K₃ PO₁, (pH 74.) 1 mM MgCl₂, 1 mMbeta-mercaptoetaol and 20% glycerol) and stored at -70° C. Assay ofspecific binding of T₃ to GC2 nuclear receptors was performed asdescribed by Samuels, et al.²³ except that T₃ binding reactions wereperformed in buffer A in the presence of 200 micrograms ml⁻¹poly(dI-dC). To assay DNA binding nuclear extracts were first incubatedwith 1 microM ¹²⁵ I-T₃ (2,200 Ci mmol⁻¹) for 20 min at 22° C. to labelthe T₃ receptors to high specific activity. Aliquots (40 microliters) ofnuclear extract were then incubated with biotinylated probes in thepresence of 200 microliters poly(dI-dC) for 40 min at 22° C. Protein-DNAcomplexes were precipitated by addition of streptavidin conjugated toagarose beads (BRL). The agarose beads were pelleted, washed three timeswith buffer A (1 ml) and assayed for ¹²⁵ I activity.

FIG. VI-3

DNase I footprinting of the rat GH enhancer element by GC2 nuclearextracts. A 16-bp protected region in the antisense strand is shown. Asecond footprint extending from position -110 to position -40 from theCAP site is also evident. Lanes A-C, Digestion of labeled GH enhancerafter incubation with GC2 nuclear extract, using 24, 12 and 4 microgramDNase I respectively. Lanes D-F, Digestion of labeled GH enhancer with24, 12 and 4 microgram of DNase I in the absence of GC2 nuclear extract.Lane G and M, Markers. The displayed sequence corresponds to that of thesense strand within the footprinted region.

FIG. VI-3 Method

The antisense strand of the growth hormone enhancer was labeled with ³²P-dATP at its 5' end using T4 polynucleotide kinase after BamHIdigestion of pGPO and treatment with calf intestinal phosphatase. Theenhancer fragment was released from pGPO by XhoI digestion and purifiedby agarose gel electrophoresis. Labeled GH enhancer fragment (1 ng, 8fmol) was incubated with 25 microliters of GC2 nuclear extractscontaining 12 fmol of specific T₃ receptor-binding activity. The DNAbinding reaction was carried out for 30 min at 22° C. in Buffer A. DNasedigestion was for 2 min at 22° C. using the above concentrations ofDNase I in a final volume of 50 microliters. The reactions were stoppedwith 20 microliters 50 mM EDTA and 1% SDS. Samples were extracted oncewith phenol-chloroform, ethanol precipitated, and analyzed byelectrophoresis on standard 10% sequencing gels.

FIG. VI-4

Binding to oligonucleotides containing 64 and 29 base pairs of 5'flanking GH sequence of rat pituitary cell T₃ and incubated with 100fmol G209-146, G186-158 or P-EGF and assayed for binding as described inFIG. VI-2. Also, hc-erb-A in vitro translation product (4 microliters)labeled with ³⁵ S-methionine was assayed for binding to theseoligonucleotides in the presence of 10 nM T₃. To prepare the hc-erb-A invitro translation product, capped mRNA transcripts of an hc-erb-Acomplementary DNA were used to program translation in a rabbitreticulocyte lysate system¹⁰. Reticulocyte lysates programmed withantisense hc-erb-A mRNA had no measurable binding activity to either ofthe GH probes (data not shown). Results are plotted as mean and standarderror of triplicate points. The experiments shown are representative ofthree experiments comparing the binding of GC2 nuclear T₃ receptors tothe two GH probes and of four experiments examining the binding of thehc-erb-A in vitro translation product.

VI. F. References Referred to in Experimental Section VI

1. Evans, R. M., Birnberg, N. C., and Rosenfeld, M. G., Proc. Natl.Acad. Sci. USA, 79:7659-7663 (1982).

2. Diamond, D. J., and Goodman, H. M., J. Molec. Biol., 181:41-62(1985).

3. Spindler, S. R., Mellon, S. H., and Baxter, J. D., J. Biol. Chem.,257:11627-11632 (1982).

4. Yaffee, B. M., and Samuels, H. H., J. Biol. Chem., 259:6284-6291(1984).

5. Larsen, P. R., Harney, J. W., and Moore, D. D., Flug, F., et al., J.Biol. Chem., 262:6373-6382 (1987).

7. Casanova, J., Copp, R. P., Janocko, L., and Samuels, H. H., J. Biol.Chem., 260:11744-11748 (1985).

8. Crew, M., and Spindler, S. R., J. Biol. Chem., 261:5018-5022 (1986).

15. Sap, J., et al., Nature, 324:635-640 (1986).

10. Weinberger, C., et al., Nature, 324:641-646 (1986).

Nelson, C., et al., Nature, 322:557-562 (1986).

12. Elsholtz, H. P., et al., Science, 234:1552-1557 (1986).

13. West, B. L., et al., Molec. Cell. Biol., 7:1193-1197 (1987).

14. Catanzaro, D. F., West, B. L., Baxter, J. D., and Reudelhuber, T.L., Molec. Endo., 1:90-96 (1987).

15. Nyborg, J. K., and Spindler, S. R., J. Biol. Chem., 261:5685-5688(1986).

16. Topol, J., Ruden, D. M., and Parker, C. S., Cell, 42:527-537 (1985).

17. Reinberg, D., Horikoshi, M., and Roeder, R. J., J. Biol. Chem.,262:3322-3330 (1987).

18. McKnight, S., and Tjian, R., Cell, 46:795-805 (1986).

19. Gorman, C. M., Moffat, L. F., and Howard, B. H., Molec. Cell. Biol.,2:1044-1051 (1982).

20. Linney, E., and Donerly, S., Cell, 35:693-699 (1983).

21. Kunkel, T., Proc. Natl. Acad. Sci. USA, 82:488-492 (1985).

22. Dingham, J. D., Lebovitz, R. M., and Roeder, R. G., Nucleic Acids.Res., 11:1475-1489 (1983).

23. Samuels, H. H., Tsai, J. S., Casanova, J., and Stanley, F., J. Clin.Invest., 54:853-865 (1974).

EXPERIMENTAL SECTION VII Identification of a novel thyroid hormonereceptor expressed in the mammalian central nervous system VII A.Summary

A complementary DNA clone derived from rat brain messenger RNA has beenisolated on the basis of homology to the human thyroid hormone receptorgene. Expression of this complementary DNA produces a high-affinitybinding protein for thyroid hormones. Sequence analysis and the mappingof this gene to a distinct human genetic locus indicate the existence ofmultiple human thyroid hormone receptors. Messenger RNA from this geneis expressed in a tissue-specific fashion with highest levels in thecentral nervous system.

VII. B. Introduction

Thyroid hormones are involved in a complex array of developmental andphysiological responses in many tissues of higher vertebrates (1). Theirnumerous and diverse effects include the regulation of importantmetabolic enzymes, hormones, and receptors (2). The actions of thyroidhormones are mediated through a nuclear receptor, which modulates theexpression of specific genes in target cells (3-5). These properties aresimilar to the interactions of steroid hormones with their receptors andare consistent with the recent observation of structural relatednessbetween steroid and thyroid hormone receptors (see Experimental SectionIII).

VII. C. Isolation of a Second Thyroid Receptor DNA

Despite the diversity of thyroid hormone action, it is generallyaccepted that thyroid hormone function occurs through a singlehigh-affinity nuclear receptor. However, the recent characterization ofthe thyroid hormone receptor as the cellular homolog of the v-erb-Aoncogene product (see Experimental Section III and ref. 7), along withthe previous identification of multiple c-erb-A genes on humanchromosomes 3 and 17 (see Experimental Section III and ref. 8), suggeststhe existence of multiple thyroid hormone receptors. To examine thepossibility that the mechanisms underlying the multiple thyroid hormoneresponses may be derived from the expression of structurally distinctthyroid hormone receptors, we have isolated a complementary DNA (cDNA)clone that encodes the product of one of these related loci.

A putative neuronal form of the thyroid hormone receptor was isolated byscreening a cDNA library prepared from rat brain messenger RNA (mRNA)with a 1500-bp fragment of the human thyroid hormone receptor cDNA. (SeeExperimental Section III which has been published as ref. 6.) From.sup.˜ 10⁶ phage, three positive clones were isolated, and the completenucleotide sequence of the largest of these, rbeA12, was determined(FIG. VII-1). The sequence is 2079 bp long and contains a long openreading frame of 1230 bp with a potential initiator methionine atnucleotide position 325 and a terminator codon at position 1554. Thisopen reading frame is preceded by a 5' untranslated region of at least320 bp that contains three short open reading frames upstream of theputative initiator methionine and encodes a protein of 410 amino acidresidues, with a calculated molecular mass of 45 kD.

VII. D. Comparison of the Second Thyroid Receptor with Other KnownThyroid Receptor Proteins

Comparison of the deduced amino acid sequence from rbeA12 with that ofthe human thyroid hormone receptor (see Experimental Section III)reveals that the two proteins have distinct amino termini (FIG. VII-2).The first 41 amino acids of the neuronal protein and the first 90 aminoacids of the human thyroid hormone receptor show no significanthomology, whereas the carboxyl terminal 367 amino acids share 75%nucleotide and 82% amino acid identities. The rat protein is morerelated to the chicken thyroid hormone receptor (7) both in predictedsize and homology, and shares 82% nucleotide and 89% amino acididentity. For reference, the chicken thyroid hormone receptor isdesignated alpha (cTR alpha) because of its homology to previouslyisolated erb-A genes (8), and the human thyroid hormone receptor isdesignated beta (hTR beta). Because the rat neuronal form is morerelated to the chicken receptor, it has been designated alpha (rTRalpha).

By analogy to the steroid hormone receptors, a cysteine-rich region inthe thyroid hormone receptor is predicted to be the DNA-binding domain(see Experimental Sections III and II; also see ref. 10). In thisregion, the rTR alpha protein has 97% amino acid identity with the cTRalpha protein and 90% amino acid identity with the hTR beta protein. Theproteins are also well conserved in the carboxyl terminal portion thatis presumed to be the hormone-binding domain, again by analogy with thesteroid receptors (see Experimental Section II; also see ref. 11). Thisregion of rTR alpha shows 94% amino acid identity with cTR alpha and 85%amino acid identity with hTR beta.

VII. E. Identification of the New Thyroid Receptor

On the basis of the sequence data, it appears that the cDNA we haveisolated encodes a protein different from the previously characterizedhuman thyroid hormone receptor (see Experimental Section III). Todemonstrate that the neuronal clone is a distinct gene product, rbeA12was used to identify human homologs by Southern blot and chromosomeanalyses. Human placenta DNA digested with various restriction enzymeswas separated on an agarose gel, transferred to nitrocellulose, andhybridized with either rat or human TR-specific probes derived fromoverlapping regions of their respective genes (FIG. VII-3). Differenthybridization patterns were revealed for all of the restriction enzymestested, which indicates that the two cDNA's represent distinct genes.The same probe from the rbeA12 was hybridized to laser-sortedchromosomes prepared from human lymphoid cells (FIG. 3C). Hybridizationwas observed only to chromosome 17, consistent with previous mappingstudies that localized c-erb-A genes to human chromosome 17 (8). Thisdistinguishes rTR alpha from hTR beta, which is found on humanchromosome 3 (see Experimental Section III).

VII. F. Expression Studies

Expression studies were performed to determine whether the rTR alphacDNA encodes a functional receptor protein. The product of the rTR alphagene was first characterized by in vitro transcription followed by invitro translation. For in vitro transcription, the EcoRI insert ofrbeA12 was linked to the bacteriophage SP6 promoter by subcloning intothe expression vector pGEM1. A second construction, rbeA12B, was createdin an attempt to increase the efficiency of translation. The 5'untranslated region up to nucleotide position 97 was deleted, whichremoved two of the three short open reading frames in this region.Transcripts synthesized with SP6 polymerase were translated in vitrowith rabbit reticulocyte lysates, and the [³⁵ S]methionine-labeledproducts were analyzed on an SDS-polyacrylamide gel (FIG. VII-4A). Fourproteins of approximately 52, 48, 35, and 33 kD were observed only whenthe sense strand was translated. The same four bands were observed forrbeA12 and rbeA12B. These translation products were then used to testthyroid hormone binding.

VII. G. Hormone Binding Studies

Thyroid hormone binding was measured with [¹²⁵I]3,5,3'-triiodo-L-thyronine (¹²⁵¹ -T₃). Only samples that contained therTR alpha specific proteins exhibited T₃ binding. Hormone affinity wasdetermined by Scatchard analysis, which gave a dissociation constant(K_(d)) of 2.9×10⁻¹¹ M (FIG. VII-4B), similar to the K_(d) observed forthe hTR beta protein (5×10⁻¹¹ M) (4, 5) and an order of magnitude lowerthan that determined for the cTR alpha protein (2.1×10⁻¹⁰ to 3.3×10⁻¹⁰M) (see Experimental Section III). The different K_(d) values obtainedmay be due to differences in the assay systems used. In competitionexperiments, the rTR alpha proteins translated in vitro showed the samecharacteristic affinities for L-T₃ and L-thyroxine (L-T₄) as the hTRbeta protein but revealed a different pattern for3,5',3'-triiodothyroacetic acid (TRIAC) (FIG. VII-4C). TRIAC competedbetter for T₃ binding with the hTR beta protein, whereas it competedabout as well at T₃ for binding to the rTR alpha protein. As with thehTR beta and cTR alpha proteins, there was no competition for T₃ bindingto the rTR alpha protein by excess aldosterone, estrogen, progesterone,testosterone, or vitamin D₁. Thus, it appears that we have isolated athyroid hormone receptor with binding properties similar to but notidentical to those of the thyroid hormone receptors previously described(see Experimental Section III and ref. 7).

VII. H. Tissue Specificity Studies

The tissue specificity of metabolic responses to thyroid hormone led usto consider that this thyroid hormone receptor might be expressed in arestricted set of tissues. Therefore, the pattern of expression of therTR alpha gene was determined by Northern blot analysis (FIG. VII-5).Total RNA isolated from various rat tissues was separated on aformaldehyde-agarose gel, transferred to nitrocellulose, and hybridizedto the same fragment of rbeA12 used for the Southern blot analysis andchromosome mapping. A 2.6-kb RNA was observed in all tissues testedexcept liver. This message is also present in pituitary and muscle andis expressed in GC, rat-1, and PC12 cell lines. Densitometric scanningindicated that the level of expression of rTR alpha was 10- to 20-foldas high in brain as in any other tissue tested. Two additional RNA's ofapproximately 5.0 and 6.0 kb are present in about equal amounts in alltissues, although they are much less abundant than the 2.6-kb message.These bands may represent precursors of the 2.6-kb message or may beproducts of a related gene.

VII. I. Discussion and Conclusions

The isolation of a second mammalian thyroid hormone receptor issurprising because previous biochemical studies have not predicted theexistence of more than a single receptor for thyroid hormones. Inretrospect, much of the clinical and physiological studies can beinterpreted as indicating the existence of multiple receptors. A form offunctional heterogeneity has been suggested by the identification ofpatients with familial thyroid hormone resistance in which peripheralresponse to thyroid hormones is lost or diminished, while neuronalfunctions are maintained (12, 13). Furthermore, severe developmentaleffects associated with low circulating thyroid hormone levels(cretinism) have been classified into types severely affecting thenervous system and those more dramatically affecting peripheralfunctions (13, 14).

In addition to demonstrating the existence of structurally distinctforms of the thyroid hormone receptor, the form that we havecharacterized is expressed at high levels in the rat central nervoussystem. Preliminary studies utilizing in situ hybridization haverevealed high levels of expression in the hippocampus, hypothalamus,cortex, and amygdala. RNA hybridization studies indicate exceptionallyhigh levels in the cerebellum as well. Although it is known that thyroidhormones play a critical role in early brain development (14), this highlevel of expression is unexpected because biochemical studies have shownthat brain has fewer thyroid hormone receptors than many other tissues(5, 16), and the adult brain is not responsive to thyroid hormone bytraditional phosphate dehydrogenase activity (17).

The second interesting result from the expression studies is that thistranscript is not present in liver, which is the tissue from whichthyroid hormone receptors usually have been isolated. This absencesuggests the existence of yet another form of the thyroid hormonereceptor. This proposal would be consistent with the data of Underwood,et al. (18), which indicates the existence of pharmacologicallydistinguishable thyroid hormone responses between liver and heart.Furthermore, data from DNA hybridization studies indicate the existenceof multiple genetic loci that hybridize with the cDNA clones for themammalian thyroid hormone receptor and suggest that there may be as manyas five different related loci (see Experimental Section III and ref.8). It seems likely that some of these loci will encode additionalfunctional molecules, which leads us to propose the existence of afamily of thyroid hormone receptors that coordinately regulateoverlapping networks of genes to control developmental and homeostaticfunction.

VII. J. Detailed Description of Figures Referred to in ExperimentalSection VII FIG. VII-1

Restriction map and nucleotide and predicted amino acid sequence ofthyroid hormone receptor cDNA from rat brain. (A) Schematicrepresentation of thyroid hormone receptor cDNA from rat brain; somecommon restriction endonuclease cleavage sites are indicated. Thehatched box indicates the predicted coding region. The 500-bp PvuIIfragment (corresponding to nucleotide positions 607 to 1113) used forthe hybridization studies is represented by the solid bar below therestriction map. (B) The complete nucleotide sequence of rbeA12 is shownwith the predicted amino acid sequence given above the long open readingframe. The three short open reading frames in the 5' untranslated regionare shown in bold type with termination codons underlined. Clone rbeA12was isolated by using the entire EcoRI insert of pheA4 (sigma) as anick-translated probe to screen .sup.˜ 10⁶ phage from a rat brain cDNAlibrary obtained from J. Arriza (19). Three positive clones wereisolated, and the complete nucleotide sequence of the largest of these,rbeA12, was determined on both strands by the chemical cleavage methodof Maxam and Gilbert (20).

FIG. VII-2

Schematic comparison of the rat thyroid hormone receptor (rTR alpha)protein with the human thyroid hormone receptor (hTR beta) and chickenthyroid hormone receptor (cTR alpha) proteins. Numbers above the boxesindicate amino acid residues; numbers inside the boxes indicate thepercent amino acid identity within the enclosed region with the rTRalpha protein. DNA designates the putative DNA-binding domain predictedby analogy with the human glucocorticoid receptor (amino acids 421 to486 of the human glucocorticoid receptor), while T₃ /T₄ designates theputative hormone-binding domain.

FIG. VII-3

Southern blot analysis and human chromosomal localization of the rTRalpha gene. Human placenta DNA was digested with various restrictionenzymes, separated on a 0.8% agarose gel, transferred to nitrocellulose,and hybridized to either a 500-bp PvuII fragment from rbeA12 (A) or a450-bp SstI fragment from hTR beta (see Experimental Section III) thatencompasses the DNA-binding region (B). Both blots were hybridized in50% formamide, 5×SSPE (0.15M NaCl, 0.01M NaH₂ PO₄, 0.001M EDTA),1×Denhardt's solution, 0.1% SDS, and salmon sperm DNA (100micrograms/ml) at 42° C., and washed in 2×SSC (standard saline citrate)and 0.1% SDS at 68° C. Sizes of lambda HindIII markers in kilobase pairsare indicated (C). Chromosome mapping of the rTR alpha gene. Humanlymphocyte chromosomes were separated by laser cytofluorometry (21) andhybridized under the same conditions as above with the 500-bp PvuIIfragment of rbeA12.

FIG. VII-4

In vitro translation and thyroid hormone binding of rTR alpha (A) rTRalpha was transcribed in vitro and translated in a rabbit reticulocytelysate. The [³⁵ S]methionine-labeled products were separated on a 7.5%SDS-polyacrylamide gel and visualized by fluorography. Lane 1, no addedRNA; lane 2, rbeA12, which contains the entire 5' untranslated region;lane 3, rbeA12B, which contains only 97 bp of 5' untranslated sequence.Sizes of protein markers: bovine serum albumin, 66.2 kD; ovalbumin, 45kD; carbonic anhydrase, 31 kD. (B) Scatchard analysis of ¹²⁵ I-T₃binding to in vitro translated rTR alpha Lysates containing in vitrotranslated rbeA12B transcripts were assayed for specific thyroidhormone-binding activity by measuring the amount of hormone bound atdifferent concentrations of ¹²⁵ I-T₃ -K_(d) =2.9×10⁻¹¹ M (C) Competitionof thyroid hormone analogs for ¹²⁵ I-T₃ binding to in vitro translatedrTR alpha. Samples from rbeA12B programmed lysates were mixed withincreasing concentrations of unlabeled thyroid hormone or analogs tocompete with labeled hormone. Specifically bound ¹²⁵ I-T₃ is plottedversus concentration of competitor compound. The same competitionpattern was observed in four separate experiments. In vitrotranscription and translation and hormone binding were performed asdescribed (22, 23). Open circles represent TRIAC; solid circlesrepresent L-T₃ ; solid triangles represent L-T₄.

FIG. VII-5

Tissue distribution of rTR alpha mRNA. Total RNA was isolated fromvarious rat tissues with guanidine thiocyanate (24), separated on a 1%agarose-formaldehyde gel, transferred to nitrocellulose, and hybridizedwith a nick-translated 500-bp PvuII fragment from rbeA12. The tissuetype and the amount of total RNA loaded are indicated above each lane. AcDNA of CHO-B, a Chinese hamster ovary cell mRNA expressed at equivalentlevels in all tissues examined (25), was used as an internal standard.Positions of 28S and 18S ribosomal RNA are indicated.

VII. K. References Referred to in Experimental Section VII

1. Wolff, E. C. and Wolff, J. in The Thyroid Gland, Pitt-Rivers, R. V.and Trotter, W. R., Eds. (Butterworths, London, 1964), vol.1,pp.237-282; Schwartz, H. L., in Molecular Basis of Thyroid HormoneAction, Oppenheimer, J. H. and Samuels, H. H., Eds. (Academic Press, NewYork, 1983), pp. 413-444.

2. Eberhardt, N. L., Apriletti, J. W., Baxter, J. B., in BiochemicalActions of Hormones, Liewack, G., Ed. (Academic Press, New York, 1980),vol. 7, pp. 311-394.

3. Oppenheimer, J. H., Koerner, D., Schwartz, H. L., Surks, M. J. J., J.Clin. Endocrinal Metabl., 35:330 (1972); Samuels, H. H. and Tsai, J. S.,Proc. Natl. Acad. Sci. U.S.A., 70:3488 (1973).

4. Tata, J. R. and Widnell, C. C., Biochem. J., 98:604 (1966); Martial,J. A., Baxter, J. D., Goodman, H. M., Seeburg, P. H., Proc. Natl. Acad.Sci. U.S.A., 74:1816 (1977); Evans, R. M., Bimberg, N. C., Rosenfeld, M.G., ibid., 79, 7659 (1982).

5. Schwartz, H. L. and Oppenheimer, J. H., Endocrinology, 103:267(1978).

6. Weinberger, C., et al., Nature, 324:641 (1986).

7. Sap, J. et al., ibid., p. 635.

8. Spurr, N. K., et al., EMBO J., 3:159 (1984); Dayton, A. J. et al.,Proc. Natl. Acad. Sci. U.S.A., 81:4495 (1984); Jhanwar, S. C. Chaganti,R. S. K., Croce, C. M., Somaric Cell Mol. Genet., 11:99 (1985).

9. Giguere, V., Hollenberg, S. M., Rosenfeld, M. G., Evans, R. M., Cell,46:645 (1986).

10. Hollenberg, S. M., Giguere, V., Segui, P., Evans, R. M., ibid, 49:39(1987).

11. Kumar, V., Green, S., Staub, A., Chambon, P., EMBO J., 5:2231(1986).

12. Menezes-Ferreira, M. M., Bil, C., Wortsman, J., Weintraub, B. D., J.Clin. Endocrinal Metab., 59:1081 (1984).

13. Burrow, G. N., in Endocrinology and Metabolism, Felig, P., Baxter,J. D., Broadhus, A. E., Frohman, L. A., Eds. (McGraw-Hill, New York,1981), pp.351 385.

14. DeGroot, L. J. and Stanbury, J. B., The Thyroid and It's Diseases(Wiley, New York, ed. 4, 1975).

15. Bayrs, J. T., Br. J. Anim. Behav., 1:144 (1953); Ford, D. H. andCramer, E. B., in Thyroid Hormones and Brain Development, Grave, G. Ed.(Raven New York 1977) pp. 1-17.

16. Oppenheimer, J. H., Schwartz, H. L., Surks, M. I., Endocrinology,95:897 (1974).

17. Barker, S. P. and Klitgaard, H. M., Am. J. Physiol., 170:81 (1952);Lee, Y. P. and Lardy, H. A., J. Biol. Chem., 240:1427 (1965).

18. Underwood, A. H. et al., Nature, 324:425 (1986).

19. The rat brain cDNA library was constructed by means ofoligo(dT)-selected RNA from whole rat brain. Moloney murine leukemiavirus (M-MuLV) reverse transcriptase (RT) was used for first strandsynthesis; second strand synthesis was with the Klenow fragment of DNApolymerase I followed by M-MuLV RT-cDNAs were treated with SI nuclease,methylated, size-fractionated on Sepharose 4B, ligated to lambda gtIIarms, and packaged. The library consists of 25×10⁶ independentrecombinants with inserts >500 bp.

20. Maxam, A. M. and Gilbert, W., Proc. Natl. Acad. Sci. U.S.A., 74:560(1977).

21. Lebo, R. V. et al., Science, 225:57 (1984).

22. Krieg, P. A. and Melton, D. A., Nucleic Acids Res., 12:7057 (1984);Hollenberg, S. M. et al., Nature, 318:635 (1985).

23. For in vitro transcription, the entire EcoRI insert of rbeA12 wascloned into pGEM1 (Promega Biotec). A second construction deleting 227bp of 5' untranslated sequence, rbeA12B, was made by inserting the T₄DNA polymerase-filled BgI-SmaI fragment of rbeA12 (nucleotide position227 to the polylinker) into the SmaI site of pGEM3. For thyroid hormonebinding, transcriptions were performed with SP6 polymerase and 5 to 10micrograms of rbeA12B linearized with SstI. Transcripts were purified byP60 chromatography and translated in 150 to 200 microliters of rabbitreticulolyte lysate (Promega Biotec) in conditions suggested by themanufacturer. Thyroid hormone binding for both the Scatchard andcompetition analyses were determined in the same manner, except thatunlabeled protein was used for the Scatchard analysis. [¹²⁵I]3,3',5:Triodothyronine (New England Nuclear, 2200 Ci/mmol, 0.3 nMfinal concentration) was mixed with rTR alpha polypeptides synthesizedin vitro (5 to 8 microliters of the 200 microliters of lysate perbinding reaction) in T₃ binding buffer at 0° C. for 2 hours in a finalvolume of 250 microliters. Specific hormone binding was determined byadding a 1000-fold excess of unlabeled hormone and assayed by countingradioactivity eluting in the excluded volume from a Sephadex G-25 fine(Pharmacia) 0.9-by 4.0-cm. column.

24. Chirgwin, J. M., Przbyla, A. F., MacDonald, R. J., Rutter, W. F.,Biochemistry, 18:5294 (1974).

25. Harpold, M. M., Evans, R. M., Salditt-Goeorgieff, M., Darnell, J.E., Cell, 17:1025 (1979).

SPECIFICATION SUMMARY

From the foregoing description, one of ordinary skill in the art canunderstand that the present invention provides substantially pure DNA'scomprised of sequences which encode proteins having the hormone-bindingand/or transcription-activating characteristics of a glucocorticoidreceptor, a mineralocorticoid receptor, or a thyroid hormone receptor.The invention also provides various plasmids containing receptorsequences which exemplify the DNA's of the invention. Exemplary plasmidsof the invention have been deposited with the American Type cultureCollection for patent purposes.

The invention is also comprised of receptor proteins, including modifiedfunctional forms thereof, expressed from the DNA's (or mRNA's) of theinvention.

In addition to novel receptor DNA, RNA and protein compositions, thepresent invention involves a bioassay for determining the functionalityof a receptor protein. It also involves two new methods for producingdesired proteins in genetically engineered cells. The first is a methodfor inducing transcription of a gene whose transcription is activated byhormones complexed with the receptors. The second is a method forengineering a cell and then increasing and controlling production of aprotein encoded by a gene whose transcription is activated by hormonescomplexed with receptor proteins.

The DNA's of the invention can be used to make the hormone receptorproteins, and functional modified forms thereof, in quantities that werenot previously possible. With the quantities of receptor available as aresult of the present invention, detailed structural analyses of theproteins can now be made by using X-ray diffraction methods to analyzereceptor crystals. In addition, adequate supplies of the receptorproteins mean that they can now be used to screen compounds forreceptor-agonists or receptor-antagonist activity. Availability of thereceptor proteins also means that they can be used in diagnostic assays.

Without departing from the spirit and scope of this invention, one ofordinary skill can make various changes and modifications to theinvention to adapt it to various usages and conditions. As such, thesechanges and modifications are properly, equitably, and intended to be,within the full range of equivalence of the following claims.

What is claimed is:
 1. A bioassay for determining whether (1) a protein(R) suspected of being a hormone receptor, or (2) a functionalengineered or modified form (r) thereof, has transcription-activatingproperties of a hormone receptor, said bioassay comprising:(a) culturingcells containing non-endogenous DNA which expresses (R) or (r) and whichcontains a DNA sequence encoding an operative hormone response elementlinked to an operative reporter gene, the culturing being conducted in aculture medium containing a known hormone (H), or an analog thereof (aH)known to have transcription activating properties of said hormone, (H),and (b) monitoring in said cells induction of the product of saidreporter gene as an indication of functional transcription-activatingbinding between hormone (H) or hormone analog (aH) with (R) or (r).
 2. Abioassay for evaluating whether compounds are functional ligands forreceptor protein (R), or a functional engineered or modified form (r)thereof, wherein said functional engineered or modified form (r) hastranscription-activating properties of receptor protein (R), saidbioassay comprising:(a) culturing cells which contain non-endogenous DNAwhich expresses (R) or a functional engineered or modified form (r)thereof, wherein said functional engineered or modified form (r) hastranscription-activating properties of (R), and which cells also containa DNA sequence encoding an operative hormone response element linked toan operative reporter gene, the culturing being conducted in culturemedium containing at least one compound (C) whose ability tofunctionally bind (R) or (r) is sought to be determined, and (b)monitoring in said cells induction of the product of said report gene asan indicator or functional binding between compound (C) and (R) or (r).3. A bioassay for determining whether a protein hastranscription-activating properties of a hormone receptor, said bioassaycomprising:culturing host cells containing reporter plasmid comprisingan operative hormone response element functionally linked to anoperative reporter gene, and expression plasmid comprising a DNAsequence encoding protein (R) or an engineered or modified form (r)thereof wherein (r) has transcription-activating properties of areceptor hormone, said culturing being done in the presence and absenceof a hormone or analog thereof, said hormone or analog being able toactivate said hormone response element in the presence of a protein thatfunctions as a receptor for said hormone or analog thereof; andmonitoring in said cells induction of reporter gene product.
 4. Abioassay according to any of claims 1, 2 or 3 wherein said host cellsare selected from the group consisting of CV-1 and COS cells.
 5. Abioassay according to claim 3 wherein said reporter and expressionplasmids also contain the origin of replication of SV-40.
 6. A bioassayaccording to claim 3 wherein said reporter and expression plasmids alsocontain a selectable marker.
 7. A bioassay according to any of claims 1,2 or 3 wherein said hormone response element is selected from the groupconsisting of mouse tumor virus long terminal repeat (MTV LTR) andmammalian growth hormone promoter, and said reporter gene is CAT.
 8. Abioassay according to any of claims 1, 2 and 3 wherein said protein (R)or said modified functional form (r) thereof is selected from the groupconsisting of steroid hormone receptors.